Allele specific modulators of P23H rhodopsin

ABSTRACT

The present embodiments provide methods, compounds, and compositions for treating, preventing, ameliorating, or slowing progression of retinitis pigmentosa (RP), such as autosomal dominant retinitis pigmentosa (AdRP) by administering a P23H rhodopsin specific inhibitor to a subject. The present embodiments provided herein are directed to compounds and compositions useful for treating, preventing, ameliorating, or slowing progression of retinitis pigmentosa (RP), such as autosomal dominant retinitis pigmentosa (AdRP). In certain embodiments, P23H rhodopsin inhibitors provided herein are allele-specific antisense compounds targeted to a P23H mutant allele that are capable of selectively inhibiting expression of P23H rhodopsin mutant protein to a greater extent than wild-type protein. In certain embodiments, administration of the allele specific antisense compounds in a subject having AdRP results in selective inhibition of P23H rhodopsin and allows the normal protein produced from the wild-type allele to maintain rod survival and function in the subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 16/553,799, filed Aug. 28, 2019, now U.S. Pat. No. 11,013,758, which is a continuation of U.S. patent application Ser. No. 15/553,876, filed Aug. 25, 2017, now U.S. Pat. No. 10,426,789, which is a U.S. National Phase under 35 U.S.C. § 371 of International Application No. PCT/US2016/019725, filed Feb. 26, 2016, which claims priority to U.S. Provisional Patent Application No. 62/121,286, filed Feb. 26, 2015, each of which is hereby incorporated by reference in its entirety into this application.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled BIOL0267WOSEQ_ST25.txt created Feb. 26, 2016, which is 60 kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

FIELD

The present embodiments provide methods, compounds, and compositions for treating, preventing, ameliorating, or slowing progression of retinitis pigmentosa (RP), such as autosomal dominant retinitis pigmentosa (AdRP) by administering a P23H rhodopsin specific inhibitor to a subject.

BACKGROUND

Retinitis pigmentosa (RP) is a broad description for pigment changes and/or damage in the retina. A hereditary form of retinitis pigmentosa called autosomal dominant retinitis pigmentosa (AdRP) is a degenerative disease that typically causes blindness by middle age. Bird A C, American journal of ophthalmology 1995; 119:543-562; Boughman J A et al. Am J Hum Genet 1980; 32:223-235; Schuster A et al. Br J Ophthalmol 2005; 89:1258-1264. AdRP is caused by abnormalities of the photoreceptors (rods and cones) or the retinal pigment epithelium (RPE) of the retina leading to progressive sight loss. AdRP patients may experience defective light to dark, dark to light adaptation or night blindness as the result of the degeneration of the peripheral visual field. AdRP results in loss of photoreceptor (rods) cells from peripheral retina and then cones from central retina.

Over 100 rhodopsin mutations have been identified in patients with AdRP. Sullivan L S et al. Invest Ophthalmol Vis Sci 2006; 47:3052-3064; Wang D Y et al. Clinica chimica acta; international journal of clinical chemistry 2005; 351:5-16. The P23H mutation is the most prevalent mutation and is present in ˜25% of AdRP and 5-15% of RP cases. Dryja T P et al. Proc Natl Acad Sci USA 1991; 88:9370-9374. Mutant rhodopsin protein such as P23H has a toxic gain-of-function that induces misfolding and disruption of normal rhodopsin protein, which leads to photoreceptor cell apoptosis. Typically, rods degenerate first, affecting low light vision. Then, cones degenerate, affecting bright light and color vision. The age of onset is variable with gradual progressive reduction in night and peripheral vision, often leading to “gun-barrel” visual field or tunnel vision. Median age of night-blindness onset is 12-14 years old. Blindness is frequent in middle ages and most rod cells are lost by age 40.

SUMMARY

The present embodiments provided herein are directed to potent, tolerable, and/or selective compounds and compositions useful for treating, preventing, ameliorating, or slowing progression of retinitis pigmentosa (RP), such as autosomal dominant retinitis pigmentosa (AdRP). In certain embodiments, P23H rhodopsin inhibitors provided herein are allele-specific antisense compounds targeted to a P23H mutant allele that are capable of selectively inhibiting expression of P23H rhodopsin mutant protein to a greater extent than wild-type protein. In certain embodiments, administration of the allele-specific antisense compounds in a subject having AdRP results in selective inhibition of P23H rhodopsin and allows the normal protein produced from the wild-type allele to maintain rod survival and function in the subject.

DETAILED DESCRIPTION

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including” as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference for the portions of the document discussed herein, as well as in their entirety.

It is understood that the sequence set forth in each SEQ ID NO in the examples contained herein is independent of any modification to a sugar moiety, an internucleoside linkage, or a nucleobase. As such, antisense compounds defined by a SEQ ID NO may comprise, independently, one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase. Antisense compounds described by ISIS number (ISIS #) indicate a combination of nucleobase sequence, chemical modification, and motif.

Unless otherwise indicated, the following terms have the following meanings:

“2′-O-methoxyethyl” (also 2′-MOE and 2′-O(CH₂)₂—OCH₃) refers to an O-methoxy-ethyl modification at the 2′ position of a sugar ring, e.g. a furanose ring. A 2′-O-methoxyethyl modified sugar is a modified sugar.

“2′-MOE nucleoside” (also 2′-O-methoxyethyl nucleoside) means a nucleoside comprising a 2′-MOE modified sugar moiety.

“2′-substituted nucleoside” means a nucleoside comprising a substituent at the 2′-position of the furanosyl ring other than H or OH. In certain embodiments, 2′ substituted nucleosides include nucleosides with bicyclic sugar modifications.

“3′ target site” refers to the nucleotide of a target nucleic acid which is complementary to the 3′-most nucleotide of a particular antisense compound.

“5′ target site” refers to the nucleotide of a target nucleic acid which is complementary to the 5′-most nucleotide of a particular antisense compound.

“5-methylcytosine” means a cytosine modified with a methyl group attached to the 5 position. A 5-methylcytosine is a modified nucleobase.

“About” means within +10% of a value. For example, if it is stated, “the compounds affected at least about 70% inhibition of P23H rhodopsin, it is implied that P23H rhodopsin levels are inhibited within a range of 60% and 80%.

“Administration” or “administering” refers to routes of introducing an antisense compound provided herein to a subject to perform its intended function. An example of a route of administration that can be used includes, but is not limited to intravitreal administration.

“Allele specific” with respect to an inhibitor refers to an inhibitor, such as an antisense compound, designed to hybridize to and/or inhibit expression of a transcript from one allele of a gene to a greater extent than the other allele of the gene.

“Amelioration” refers to a lessening of at least one indicator, sign, or symptom of an associated disease, disorder, or condition. In certain embodiments, amelioration includes a delay or slowing in the progression of one or more indicators of a condition or disease. The severity of indicators may be determined by subjective or objective measures, which are known to those skilled in the art.

“Animal” refers to a human or non-human animal, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees.

“Antisense activity” means any detectable or measurable activity attributable to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity is a decrease in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid.

“Antisense compound” means an oligomeric compound that is is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding. Examples of antisense compounds include single-stranded and double-stranded compounds, such as, antisense oligonucleotides, siRNAs, shRNAs, ssRNAs, and occupancy-based compounds.

“Antisense inhibition” means reduction of target nucleic acid levels in the presence of an antisense compound complementary to a target nucleic acid compared to target nucleic acid levels in the absence of the antisense compound.

“Antisense mechanisms” are all those mechanisms involving hybridization of a compound with target nucleic acid, wherein the outcome or effect of the hybridization is either target degradation or target occupancy with concomitant stalling of the cellular machinery involving, for example, transcription or splicing.

“Antisense oligonucleotide” means a single-stranded oligonucleotide having a nucleobase sequence that permits hybridization to a corresponding region or segment of a target nucleic acid.

“Base complementarity” refers to the capacity for the precise base pairing of nucleobases of an antisense oligonucleotide with corresponding nucleobases in a target nucleic acid (i.e., hybridization), and is mediated by Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen binding between corresponding nucleobases.

“Bicyclic sugar moiety” means a modified sugar moiety comprising a 4 to 7 membered ring (including but not limited to a furanosyl) comprising a bridge connecting two atoms of the 4 to 7 membered ring to form a second ring, resulting in a bicyclic structure. In certain embodiments, the 4 to 7 membered ring is a sugar ring. In certain embodiments the 4 to 7 membered ring is a furanosyl. In certain such embodiments, the bridge connects the 2′-carbon and the 4′-carbon of the furanosyl.

“Cap structure” or “terminal cap moiety” means chemical modifications, which have been incorporated at either terminus of an antisense compound.

“cEt” or “constrained ethyl” means a bicyclic sugar moiety comprising a bridge connecting the 4′-carbon and the 2′-carbon, wherein the bridge has the formula: 4′-CH(CH₃)—O-2′.

“Constrained ethyl nucleoside” (also cEt nucleoside) means a nucleoside comprising a bicyclic sugar moiety comprising a 4′-CH(CH₃)—O-2′ bridge.

“P23H rhodopsin” means any nucleic acid or protein of P23H rhodopsin. “P23H rhodopsin nucleic acid” means any nucleic acid encoding P23H rhodopsin. For example, in certain embodiments, a P23H rhodopsin nucleic acid includes a DNA sequence encoding P23H rhodopsin, an RNA sequence transcribed from DNA encoding P23H rhodopsin (including genomic DNA comprising introns and exons), and an mRNA sequence encoding P23H rhodopsin. “P23H rhodopsin mRNA” means an mRNA encoding a P23H rhodopsin protein.

“P23H rhodopsin specific inhibitor” refers to any agent capable of specifically inhibiting P23H rhodopsin RNA and/or P23H rhodopsin protein expression or activity at the molecular level. For example, P23H rhodopsin specific inhibitors include nucleic acids (including antisense compounds), peptides, antibodies, small molecules, and other agents capable of inhibiting the expression of P23H rhodopsin RNA and/or P23H rhodopsin protein.

“Chemically distinct region” refers to a region of an antisense compound that is in some way chemically different than another region of the same antisense compound. For example, a region having 2′-O-methoxyethyl nucleotides is chemically distinct from a region having nucleotides without 2′-O-methoxyethyl modifications.

“Chimeric antisense compounds” means antisense compounds that have at least 2 chemically distinct regions, each position having a plurality of subunits.

“Complementarity” means the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid.

“Comprise,” “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

“Contiguous nucleobases” means nucleobases immediately adjacent to each other.

“Deoxyribonucleotide” means a nucleotide having a hydrogen at the 2′ position of the sugar portion of the nucleotide. Deoxyribonucleotides may be modified with any of a variety of substituents.

“Designing” or “Designed to” refer to the process of designing an oligomeric compound that specifically hybridizes with a selected nucleic acid molecule.

“Effective amount” means the amount of active pharmaceutical agent sufficient to effectuate a desired physiological outcome in an individual in need of the agent. The effective amount may vary among individuals depending on the health and physical condition of the individual to be treated, the taxonomic group of the individuals to be treated, the formulation of the composition, assessment of the individual's medical condition, and other relevant factors.

“Efficacy” means the ability to produce a desired effect.

“Expression” includes all the functions by which a gene's coded information is converted into structures present and operating in a cell. Such structures include, but are not limited to the products of transcription and translation.

“Fully complementary” or “100% complementary” means each nucleobase of a first nucleic acid has a complementary nucleobase in a second nucleic acid. In certain embodiments, a first nucleic acid is an antisense compound and a target nucleic acid is a second nucleic acid.

“Gapmer” means a chimeric antisense compound in which an internal region having a plurality of nucleosides that support RNase H cleavage is positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions. The internal region may be referred to as the “gap” and the external regions may be referred to as the “wings.”

“Hybridization” means the annealing of complementary nucleic acid molecules. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an antisense compound and a nucleic acid target. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an antisense oligonucleotide and a nucleic acid target.

“Identifying an animal having, or at risk for having, a disease, disorder and/or condition” means identifying an animal having been diagnosed with the disease, disorder and/or condition or identifying an animal predisposed to develop the disease, disorder and/or condition. Such identification may be accomplished by any method including evaluating an individual's medical history and standard clinical tests or assessments.

“Immediately adjacent” means there are no intervening elements between the immediately adjacent elements.

“Individual” means a human or non-human animal selected for treatment or therapy.

“Inhibiting the expression or activity” refers to a reduction, blockade of the expression or activity and does not necessarily indicate a total elimination of expression or activity.

“Internucleoside linkage” refers to the chemical bond between nucleosides.

“Lengthened” antisense oligonucleotides are those that have one or more additional nucleosides relative to an antisense oligonucleotide disclosed herein.

“Linked deoxynucleoside” means a nucleic acid base (A, G, C, T, U) substituted by deoxyribose linked by a phosphate ester to form a nucleotide.

“Linked nucleosides” means adjacent nucleosides linked together by an internucleoside linkage.

“Mismatch” or “non-complementary nucleobase” refers to the case when a nucleobase of a first nucleic acid is not capable of pairing with the corresponding nucleobase of a second or target nucleic acid.

“Modified internucleoside linkage” refers to a substitution or any change from a naturally occurring internucleoside bond (i.e. a phosphodiester internucleoside bond).

“Modified nucleobase” means any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil. An “unmodified nucleobase” means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).

“Modified nucleoside” means a nucleoside having, independently, a modified sugar moiety and/or modified nucleobase.

“Modified nucleotide” means a nucleotide having, independently, a modified sugar moiety, modified internucleoside linkage, or modified nucleobase.

“Modified oligonucleotide” means an oligonucleotide comprising at least one modified internucleoside linkage, a modified sugar, and/or a modified nucleobase.

“Modified sugar” means substitution and/or any change from a natural sugar moiety.

“Modulating” refers to changing or adjusting a feature in a cell, tissue, organ or organism. For example, modulating P23H rhodopsin mRNA can mean to increase or decrease the level of P23H rhodopsin mRNA and/or P23H rhodopsin protein in a cell, tissue, organ or organism. A “modulator” effects the change in the cell, tissue, organ or organism. For example, a P23H rhodopsin antisense compound can be a modulator that decreases the amount of P23H rhodopsin mRNA and/or P23H rhodopsin protein in a cell, tissue, organ or organism.

“Monomer” refers to a single unit of an oligomer. Monomers include, but are not limited to, nucleosides and nucleotides, whether naturally occurring or modified.

“Motif” means the pattern of unmodified and modified nucleosides in an antisense compound.

“Natural sugar moiety” means a sugar moiety found in DNA (2′-H) or RNA (2′-OH).

“Naturally occurring internucleoside linkage” means a 3′ to 5′ phosphodiester linkage.

“Non-complementary nucleobase” refers to a pair of nucleobases that do not form hydrogen bonds with one another or otherwise support hybridization.

“Nucleic acid” refers to molecules composed of monomeric nucleotides. A nucleic acid includes, but is not limited to, ribonucleic acids (RNA), deoxyribonucleic acids (DNA), single-stranded nucleic acids, and double-stranded nucleic acids.

“Nucleobase” means a heterocyclic moiety capable of pairing with a base of another nucleic acid.

“Nucleobase complementarity” refers to a nucleobase that is capable of base pairing with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In certain embodiments, complementary nucleobase refers to a nucleobase of an antisense compound that is capable of base pairing with a nucleobase of its target nucleic acid. For example, if a nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at that nucleobase pair.

“Nucleobase sequence” means the order of contiguous nucleobases independent of any sugar, linkage, and/or nucleobase modification.

“Nucleoside” means a nucleobase linked to a sugar.

“Nucleoside mimetic” includes those structures used to replace the sugar or the sugar and the base and not necessarily the linkage at one or more positions of an oligomeric compound such as for example nucleoside mimetics having morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclo or tricyclo sugar mimetics, e.g., non furanose sugar units. Nucleotide mimetic includes those structures used to replace the nucleoside and the linkage at one or more positions of an oligomeric compound such as for example peptide nucleic acids or morpholinos (morpholinos linked by —N(H)—C(═O)—O— or other non-phosphodiester linkage). Sugar surrogate overlaps with the slightly broader term nucleoside mimetic but is intended to indicate replacement of the sugar unit (furanose ring) only. The tetrahydropyranyl rings provided herein are illustrative of an example of a sugar surrogate wherein the furanose sugar group has been replaced with a tetrahydropyranyl ring system. “Mimetic” refers to groups that are substituted for a sugar, a nucleobase, and/or internucleoside linkage. Generally, a mimetic is used in place of the sugar or sugar-internucleoside linkage combination, and the nucleobase is maintained for hybridization to a selected target.

“Nucleotide” means a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside.

“Oligomeric compound” means a polymer of linked monomeric subunits which is capable of hybridizing to at least a region of a nucleic acid molecule.

“Oligonucleoside” means an oligonucleotide in which the internucleoside linkages do not contain a phosphorus atom.

“Oligonucleotide” means a polymer of linked nucleosides each of which can be modified or unmodified, independent one from another.

“Pharmaceutical composition” means a mixture of substances suitable for administering to an individual. For example, a pharmaceutical composition may comprise one or more active pharmaceutical agents and a sterile aqueous solution.

“Pharmaceutically acceptable salts” means physiologically and pharmaceutically acceptable salts of antisense compounds, i.e., salts that retain the desired biological activity of the parent oligonucleotide and do not impart undesired toxicological effects thereto.

“Phosphorothioate linkage” means a linkage between nucleosides where the phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. A phosphorothioate linkage is a modified internucleoside linkage.

“Portion” means a defined number of contiguous (i.e., linked) nucleobases of a nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of an antisense compound

“Prevent” refers to delaying or forestalling the onset, development or progression of a disease, disorder, or condition for a period of time from minutes to indefinitely. Prevent also means reducing the risk of developing a disease, disorder, or condition.

“Prophylactically effective amount” refers to an amount of a pharmaceutical agent that provides a prophylactic or preventative benefit to an animal.

“Region” is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic.

“Ribonucleotide” means a nucleotide having a hydroxy at the 2′ position of the sugar portion of the nucleotide. Ribonucleotides may be modified with any of a variety of substituents.

“Segments” are defined as smaller or sub-portions of regions within a target nucleic acid.

“Selective” with respect to an effect refers to a greater effect on one thing over another by any quantitative extent or fold-difference. For example, an antisense compound that is “selective” for P23H rhodopsin or “selectively” targets or inhibits P23H rhodopsin, reduces expression of the P23H rhodopsin allele to a greater extent than the wild-type allele.

“Side effects” means physiological disease and/or conditions attributable to a treatment other than the desired effects. In certain embodiments, side effects include injection site reactions, liver function test abnormalities, renal function abnormalities, liver toxicity, renal toxicity, central nervous system abnormalities, myopathies, and malaise. For example, increased aminotransferase levels in serum may indicate liver toxicity or liver function abnormality. For example, increased bilirubin may indicate liver toxicity or liver function abnormality.

“Sites,” as used herein, are defined as unique nucleobase positions within a target nucleic acid.

“Slows progression” means decrease in the development of the said disease.

“Specifically hybridizable” refers to an antisense compound having a sufficient degree of complementarity between an antisense oligonucleotide and a target nucleic acid to induce a desired effect, while exhibiting minimal or no effects on non-target nucleic acids under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays and therapeutic treatments. “Stringent hybridization conditions” or “stringent conditions” refer to conditions under which an oligomeric compound will hybridize to its target sequence, but to a minimal number of other sequences.

“Specifically inhibit” a target nucleic acid means to reduce or block expression of the target nucleic acid while exhibiting fewer, minimal, or no effects on non-target nucleic acids and does not necessarily indicate a total elimination of the target nucleic acid's expression.

“Subject” means a human or non-human animal selected for treatment or therapy.

“Target” refers to a protein, the modulation of which is desired.

“Target gene” refers to a gene encoding a target.

“Targeting” means the process of design and selection of an antisense compound that will specifically hybridize to a target nucleic acid and induce a desired effect.

“Target nucleic acid,” “target RNA,” “target RNA transcript” and “nucleic acid target” all mean a nucleic acid capable of being targeted by antisense compounds.

“Target region” means a portion of a target nucleic acid to which one or more antisense compounds is targeted.

“Target segment” means the sequence of nucleotides of a target nucleic acid to which an antisense compound is targeted. “5′ target site” refers to the 5′-most nucleotide of a target segment. “3′ target site” refers to the 3′-most nucleotide of a target segment.

“Therapeutically effective amount” means an amount of a pharmaceutical agent that provides a therapeutic benefit to an individual.

“Treat” refers to administering a pharmaceutical composition to an animal in order to effect an alteration or improvement of a disease, disorder, or condition in the animal. In certain embodiments, one or more pharmaceutical compositions can be administered to the animal.

“Unmodified” nucleobases mean the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).

“Unmodified nucleotide” means a nucleotide composed of naturally occurring nucleobases, sugar moieties, and internucleoside linkages. In certain embodiments, an unmodified nucleotide is an RNA nucleotide (i.e. β-D-ribonucleosides) or a DNA nucleotide (i.e. β-D-deoxyribonucleoside).

CERTAIN EMBODIMENTS

Certain embodiments provide methods, compounds and compositions for inhibiting or selectively inhibiting P23H rhodopsin expression.

Certain embodiments provide antisense compounds targeted to a P23H rhodopsin nucleic acid. In certain embodiments, the human mutant P23H rhodopsin nucleic acid has a C to A substitution at nucleotide 163 of GENBANK Accession No. NM_000539.3 and is incorporated herein as SEQ ID NO: 2. In certain embodiments, the human mutant P23H rhodopsin nucleic acid has a C to A substitution in codon 23 (exon 1) of a human rhodopsin gene having the sequence of GENBANK Accession No. NM_000539.3. In certain embodiments, the antisense compound is a single-stranded oligonucleotide.

Certain embodiments provide an antisense compound comprising a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 11-64. In certain embodiments, the antisense compound is a single-stranded oligonucleotide.

Certain embodiments provide an antisense compound comprising a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising at least 9 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 11-64. In certain embodiments, the antisense compound is a single-stranded oligonucleotide.

Certain embodiments provide an antisense compound comprising a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising at least 10 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 11-64. In certain embodiments, the antisense compound is a single-stranded oligonucleotide.

Certain embodiments provide an antisense compound comprising a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising at least 11 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 11-64. In certain embodiments, the antisense compound is a single-stranded oligonucleotide.

Certain embodiments provide an antisense compound comprising a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising at least 12 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 11-64. In certain embodiments, the antisense compound is a single-stranded oligonucleotide.

Certain embodiments provide an antisense compound comprising a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 11-64. In certain embodiments, the antisense compound is a single-stranded oligonucleotide.

Certain embodiments provide an antisense compound comprising a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 11-64. In certain embodiments, the antisense compound is a single-stranded oligonucleotide.

In certain embodiments, a compound comprises or consists of a modified oligonucleotide consisting of 8 to 80 linked nucleosides having at least an 8, 9, 10, 11, 12, 13, 14, 15, or 16 contiguous nucleobase portion complementary to an equal length portion within nucleotides 157-174, 157-174, 157-171, 157-172, or 159-174 of SEQ ID NO: 2.

In certain embodiments, a compound comprises a modified oligonucleotide consisting of 8 to 80 linked nucleosides complementary within nucleotides 157-174, 157-171, 157-172, or 159-174 of SEQ ID NO: 2.

In certain embodiments, a compound comprises or consists of a modified oligonucleotide consisting of 10 to 30 linked nucleosides complementary within nucleotides 157-174, 157-174, 157-174, 157-171, 157-172, or 159-174 of SEQ ID NO: 2.

In certain embodiments, a compound comprises or consists of a modified oligonucleotide consisting of 8 to 80 linked nucleosides having a nucleobase sequence comprising at least an 8, 9, 10, 11, 12, 13, 14, 15, or 16 contiguous nucleobase portion any one of SEQ ID NOs: 15, 21, 29, or 64.

In certain embodiments, a compound comprises a modified oligonucleotide consisting of 10 to 30 linked nucleosides having a nucleobase sequence comprising at least 8 contiguous nucleobases of any one of SEQ ID NOs: 15, 21, 29, or 64.

In certain embodiments, a compound comprises a modified oligonucleotide consisting of 10 to 30 linked nucleosides having a nucleobase sequence comprising at least 9 contiguous nucleobases of any one of SEQ ID NOs: 15, 21, 29, or 64.

In certain embodiments, a compound comprises a modified oligonucleotide consisting of 10 to 30 linked nucleosides having a nucleobase sequence comprising at least 10 contiguous nucleobases of any one of SEQ ID NOs: 15, 21, 29, or 64.

In certain embodiments, a compound comprises a modified oligonucleotide consisting of 10 to 30 linked nucleosides having a nucleobase sequence comprising at least 11 contiguous nucleobases of any one of SEQ ID NOs: 15, 21, 29, or 64.

In certain embodiments, a compound comprises a modified oligonucleotide consisting of 10 to 30 linked nucleosides having a nucleobase sequence comprising at least 12 contiguous nucleobases of any one of SEQ ID NOs: 15, 21, 29, or 64.

In certain embodiments, a compound comprises or consists of a modified oligonucleotide consisting of 10 to 30 linked nucleosides having a nucleobase sequence comprising any one of SEQ ID NOs: 15, 21, 29, or 64.

In certain embodiments, a compound comprises or consists of a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ ID NOs: 15, 21, 29, or 64.

In certain embodiments, a modified oligonucleotide targeted to P23H rhodopsin is ISIS 564426, ISIS 664844, ISIS 664867, or ISIS 664884. Out of over 400 antisense oligonucleotides that were screened as described in the Examples section below, ISIS 564426, ISIS 664844, ISIS 664867, and ISIS 664884 emerged as the top lead compounds. In particular, ISIS 664844 exhibited the best combination of properties in terms of potency, tolerability, and selectivity for P23H rhodopsin out of over 400 antisense oligonucleotides.

In certain embodiments, any of the foregoing compounds or oligonucleotides comprises at least one modified internucleoside linkage, at least one modified sugar, and/or at least one modified nucleobase.

In certain embodiments, any of the foregoing compounds or oligonucleotides comprises at least one modified sugar. In certain embodiments, at least one modified sugar comprises a 2′-O-methoxyethyl group.

In certain embodiments, at least one modified sugar is a bicyclic sugar, such as a 4′-CH(CH₃)—O-2′ group, a 4′-CH₂—O-2′ group, or a 4′-(CH₂)₂—O-2′group.

In certain embodiments, the modified oligonucleotide comprises at least one modified internucleoside linkage, such as a phosphorothioate internucleoside linkage.

In certain embodiments, any of the foregoing compounds or oligonucleotides comprises at least one modified nucleobase, such as 5-methylcytosine.

In certain embodiments, any of the foregoing compounds or oligonucleotides comprises:

-   -   a gap segment consisting of linked deoxynucleosides;     -   a 5′ wing segment consisting of linked nucleosides; and     -   a 3′ wing segment consisting of linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment and wherein each nucleoside of each wing segment comprises a modified sugar. In certain embodiments, the oligonucleotide consists of 10 to 30 linked nucleosides having a nucleobase sequence comprising the sequence recited in SEQ ID NO: 15, 44, or 52.

In certain embodiments, a compound comprises or consists of a modified oligonucleotide consisting of 10 to 30 linked nucleosides having a nucleobase sequence comprising any one of SEQ ID NOs: 11-64, wherein the modified oligonucleotide comprises:

-   -   a gap segment consisting of linked deoxynucleosides;     -   a 5′ wing segment consisting of linked nucleosides; and     -   a 3′ wing segment consisting of linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.

In certain embodiments, a compound comprises or consists of a modified oligonucleotide consisting of 10 to 30 linked nucleosides having a nucleobase sequence comprising any one of SEQ ID NOs: 15, 21, 29, or 64, wherein the modified oligonucleotide comprises:

-   -   a gap segment consisting of linked deoxynucleosides;     -   a 5′ wing segment consisting of linked nucleosides; and     -   a 3′ wing segment consisting of linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.

In certain embodiments, a compound comprises or consists of a modified oligonucleotide consisting of 16 to 30 linked nucleosides having a nucleobase sequence comprising the sequence recited in SEQ ID NO: 15, wherein the modified oligonucleotide comprises:

-   -   a gap segment consisting of ten linked deoxynucleosides;     -   a 5′ wing segment consisting of three linked nucleosides; and     -   a 3′ wing segment consisting of three linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment; wherein each nucleoside of each wing segment comprises a cEt sugar; wherein each internucleoside linkage is a phosphorothioate linkage; and wherein each cytosine is a 5-methylcytosine.

In certain embodiments, a compound comprises or consists of a modified oligonucleotide consisting of 16 linked nucleosides having a nucleobase sequence consisting of the sequence recited in SEQ ID NO: 15, wherein the modified oligonucleotide comprises:

-   -   a gap segment consisting of ten linked deoxynucleosides;     -   a 5′ wing segment consisting of three linked nucleosides; and     -   a 3′ wing segment consisting of three linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment; wherein each nucleoside of each wing segment comprises a cEt sugar; wherein each internucleoside linkage is a phosphorothioate linkage; and wherein each cytosine is a 5-methylcytosine.

In certain embodiments, a compound comprises or consists of a modified oligonucleotide consisting of 16 to 30 linked nucleosides having a nucleobase sequence comprising the sequence recited in SEQ ID NO: 64, wherein the modified oligonucleotide comprises:

-   -   a gap segment consisting of nine linked deoxynucleosides;     -   a 5′ wing segment consisting of four linked nucleosides; and     -   a 3′ wing segment consisting of three linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment; wherein the 5′ wing segment comprises a cEt sugar, a cEt sugar, a cEt sugar, and a 2′-flouro sugar in the 5′ to 3′ direction; wherein each nucleoside of the 3′ wing segment comprises a cEt sugar; wherein each internucleoside linkage is a phosphorothioate linkage; and wherein each cytosine is a 5-methylcytosine.

In certain embodiments, a compound comprises or consists of a modified oligonucleotide consisting of 16 linked nucleosides having a nucleobase sequence consisting of the sequence recited in SEQ ID NO: 64, wherein the modified oligonucleotide comprises:

-   -   a gap segment consisting of nine linked deoxynucleosides;     -   a 5′ wing segment consisting of four linked nucleosides; and     -   a 3′ wing segment consisting of three linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment; wherein the 5′ wing segment comprises a cEt sugar, a cEt sugar, a cEt sugar, and a 2′-flouro sugar in the 5′ to 3′ direction; wherein each nucleoside of the 3′ wing segment comprises a cEt sugar; wherein each internucleoside linkage is a phosphorothioate linkage; and wherein each cytosine is a 5-methylcytosine.

In certain embodiments, a compound comprises or consists of a modified oligonucleotide consisting of 16 to 30 linked nucleosides having a nucleobase sequence comprising the sequence recited in SEQ ID NO: 21, wherein the modified oligonucleotide comprises:

-   -   a gap segment consisting of ten linked deoxynucleosides;     -   a 5′ wing segment consisting of two linked nucleosides; and     -   a 3′ wing segment consisting of four linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment; wherein each nucleoside of the 5′ wing segment comprises a cEt sugar; wherein the 3′ wing segment comprises a cEt sugar, a 2′-O-methoxyethyl sugar, a cEt sugar, and a 2′-O-methoxyethyl sugar in the 5′ to 3′ direction; wherein each internucleoside linkage is a phosphorothioate linkage; and wherein each cytosine is a 5-methylcytosine.

In certain embodiments, a compound comprises or consists of a modified oligonucleotide consisting of 16 linked nucleosides having a nucleobase sequence consisting of the sequence recited in SEQ ID NO: 21, wherein the modified oligonucleotide comprises:

-   -   a gap segment consisting of ten linked deoxynucleosides;     -   a 5′ wing segment consisting of two linked nucleosides; and     -   a 3′ wing segment consisting of four linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment; wherein each nucleoside of the 5′ wing segment comprises a cEt sugar; wherein the 3′ wing segment comprises a cEt sugar, a 2′-O-methoxyethyl sugar, a cEt sugar, and a 2′-O-methoxyethyl sugar in the 5′ to 3′ direction; wherein each internucleoside linkage is a phosphorothioate linkage; and wherein each cytosine is a 5-methylcytosine.

In certain embodiments, a compound comprises or consists of a modified oligonucleotide consisting of 15 to 30 linked nucleosides having a nucleobase sequence comprising the sequence recited in SEQ ID NO: 29, wherein the modified oligonucleotide comprises:

-   -   a gap segment consisting of ten linked deoxynucleosides;     -   a 5′ wing segment consisting of two linked nucleosides; and     -   a 3′ wing segment consisting of three linked nucleosides;     -   wherein the gap segment is positioned between the 5′ wing         segment and the 3′ wing segment; wherein each nucleoside of the         5′ wing segment comprises a cEt sugar; wherein the 3′ wing         segment comprises a cEt sugar, a 2′-O-methoxyethyl sugar, and a         cEt sugar in the 5′ to 3′ direction; wherein each         internucleoside linkage is a phosphorothioate linkage; and         wherein each cytosine is a 5-methylcytosine.

In certain embodiments, a compound comprises or consists of a modified oligonucleotide consisting of 15 linked nucleosides having a nucleobase sequence consisting of the sequence recited in SEQ ID NO: 29, wherein the modified oligonucleotide comprises:

-   -   a gap segment consisting of ten linked deoxynucleosides;     -   a 5′ wing segment consisting of two linked nucleosides; and     -   a 3′ wing segment consisting of three linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment; wherein each nucleoside of the 5′ wing segment comprises a cEt sugar; wherein the 3′ wing segment comprises a cEt sugar, a 2′-O-methoxyethyl sugar, and a cEt sugar in the 5′ to 3′ direction; wherein each internucleoside linkage is a phosphorothioate linkage; and wherein each cytosine is a 5-methylcytosine.

In any of the foregoing embodiments, the compound or oligonucleotide can be at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary to a nucleic acid encoding P23H rhodopsin.

In any of the foregoing embodiments, the antisense compound can be a single-stranded oligonucleotide. In certain embodiments, the compound comprises deoxyribonucleotides.

In any of the foregoing embodiments, the antisense compound can be double-stranded. In certain embodiments, a compound comprises ribonucleotides.

In certain embodiments, compounds are capable of selectively targeting or inhibiting expression of the Rhodopsin P23H mutant allele. In certain embodiments, compounds have at least about a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or 20-fold selectivity for inhibiting expression of the Rhodopsin P23H mutant allele over the wild-type allele.

In certain embodiments, compounds or compositions provided herein comprise a salt of the modified oligonucleotide. In certain embodiments, the salt is a sodium salt. In certain embodiments, the salt is a potassium salt.

Certain embodiments provide a composition comprising the compound of any of the aforementioned embodiments or salt thereof and at least one of a pharmaceutically acceptable carrier or diluent. In certain embodiments, the composition has a viscosity less than about 40 centipoise (cP), less than about 30 centipose (cP), less than about 20 centipose (cP), less than about 15 centipose (cP), or less than about 10 centipose (cP). In certain embodiments, the composition having any of the aforementioned viscosities comprises a compound provided herein at a concentration of about 100 mg/mL, about 125 mg/mL, about 150 mg/mL, about 175 mg/mL, about 200 mg/mL, about 225 mg/mL, about 250 mg/mL, about 275 mg/mL, or about 300 mg/mL. In certain embodiments, the composition having any of the aforementioned viscosities and/or compound concentrations has a temperature of room temperature or about 20° C., about 21° C., about 22° C., about 23° C., about 24° C., about 25° C., about 26° C., about 27° C., about 28° C., about 29° C., or about 30° C.

Certain Indications

Certain embodiments provided herein relate to methods of treating, preventing, ameliorating, or slowing progression of a disease associated with P23H rhodopsin in a subject by administration of a P23H rhodopsin specific inhibitor, such as an antisense compound targeted to P23H rhodopsin. In certain embodiments, the inhibitor is allele-specific for P23H Rhodospin and selectively inhibits expression of P23H rhodopsin over wild-type rhodopsin. Examples of diseases associated with P23H rhodopsin treatable, preventable, and/or ameliorable with the methods provided herein include retinitis pigmentosa (RP), such as autosomal dominant retinitis pigmentosa (AdRP).

In certain embodiments, a method of treating, preventing, ameliorating, or slowing progression of retinitis pigmentosa (RP) or autosomal dominant retinitis pigmentosa (AdRP) in a subject comprises administering to the subject a P23H rhodopsin specific inhibitor, thereby treating, preventing, ameliorating, or slowing progression of retinitis pigmentosa (RP) or autosomal dominant retinitis pigmentosa (AdRP) in the subject. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound targeted to P23H rhodopsin, such as an antisense oligonucleotide targeted to P23H rhodopsin. In certain embodiments, the antisense compound is allele-specific for P23H Rhodospin and selectively inhibits expression of P23H rhodopsin over wild-type rhodopsin. In certain embodiments, the P23H rhodopsin specific inhibitor is a compound comprising or consisting of a modified oligonucleotide consisting of 8 to 80 linked nucleosides complementary within nucleotides 157-174, 157-171, 157-172, or 159-174 of SEQ ID NO: 2. In certain embodiments, the P23H rhodopsin specific inhibitor is a compound comprising or consisting of a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 11-64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 11-64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 11-64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide consisting of 10 to 30 linked nucleosides having a nucleobase sequence comprising at least 8, 9, 10, 11, or 12 contiguous nucleobases of any one of SEQ ID NOs: 15, 21, 29, or 64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide consisting of 10 to 30 linked nucleosides having a nucleobase sequence comprising any one of SEQ ID NOs: 15, 21, 29, or 64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ ID NOs: 15, 21, 29, or 64. In certain embodiments, the P23H rhodopsin specific inhibitor is ISIS 564426, ISIS 664844, ISIS 664867, or ISIS 664884. In any of the foregoing embodiments, the antisense compound can be a single-stranded oligonucleotide. In certain embodiments, the antisense compound is administered to the subject by intravitreally such as by intravitreal injection. In certain embodiments, administering the antisense compound improves, preserves, or prevents worsening of visual function; visual field; photoreceptor cell function; electroretinogram (ERG) response such as full field ERG measuring retina wide function, dark adapted ERG measuring scotopic rod function, or light adapted ERG measuring photopic cone function; visual acuity; and/or vision-related quality of life. In certain embodiments, administering the antisense compound inhibits, prevents, or delays progression of photoreceptor cell loss and/or deterioration of the retina outer nuclear layer (ONL). In certain embodiments, the subject is identified as having the P23H rhodopsin mutant allele.

In certain embodiments, a method of inhibiting expression of P23H rhodopsin in a subject having a P23H rhodopsin mutant allele comprises administering a P23H rhodopsin specific inhibitor to the subject, thereby inhibiting expression of P23H rhodopsin in the subject. In certain embodiments, administering the inhibitor inhibits expression of P23H rhodopsin in the eye, retina, peripheral retina, rod photoreceptors, and/or cones. In certain embodiments, the subject has, or is at risk of having retinitis pigmentosa (RP), such as autosomal dominant retinitis pigmentosa (AdRP). In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound allele-specific for P23H Rhodospin that selectively inhibits expression of P23H rhodopsin over wild-type rhodopsin. In certain embodiments, the P23H rhodopsin specific inhibitor is a compound comprising or consisting of a modified oligonucleotide consisting of 8 to 80 linked nucleosides complementary within nucleotides 157-174, 157-171, 157-172, or 159-174 of SEQ ID NO: 2. In certain embodiments, the P23H rhodopsin specific inhibitor is a compound comprising or consisting of a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 11-64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 11-64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 11-64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide consisting of 10 to 30 linked nucleosides having a nucleobase sequence comprising at least 8, 9, 10, 11, or 12 contiguous nucleobases of any one of SEQ ID NOs: 15, 21, 29, or 64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide consisting of 10 to 30 linked nucleosides having a nucleobase sequence comprising any one of SEQ ID NOs: 15, 21, 29, or 64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ ID NOs: 15, 21, 29, or 64. In certain embodiments, the P23H rhodopsin specific inhibitor is ISIS 564426, ISIS 664844, ISIS 664867, or ISIS 664884. In any of the foregoing embodiments, the antisense compound can be a single-stranded oligonucleotide. In certain embodiments, the antisense compound is administered to the subject by intravitreally such as by intravitreal injection.

In certain embodiments, a method of improving or preserving visual function, visual field, photoreceptor cell function, ERG response, or visual acuity in a subject having a P23H rhodopsin mutant allele or having retinitis pigmentosa (RP), such as autosomal dominant retinitis pigmentosa (AdRP), comprises administering a P23H rhodopsin specific inhibitor to the subject. In certain embodiments, a method of inhibiting, preventing, or delaying progression of photoreceptor cell loss and/or deterioration of the retina outer nuclear layer (ONL) in a subject having a P23H rhodopsin mutant allele or having retinitis pigmentosa (RP), such as autosomal dominant retinitis pigmentosa (AdRP), comprises administering a P23H rhodopsin specific inhibitor to the subject. In certain embodiments, the inhibitor is an antisense compound targeted to P23H rhodopsin. In certain embodiments, the antisense compound is allele-specific for P23H Rhodospin and selectively inhibits expression of P23H rhodopsin over wild-type rhodopsin. In certain embodiments, the P23H rhodopsin specific inhibitor is a compound comprising or consisting of a modified oligonucleotide consisting of 8 to 80 linked nucleosides complementary within nucleotides 157-174, 157-171, 157-172, or 159-174 of SEQ ID NO: 2. In certain embodiments, the P23H rhodopsin specific inhibitor is a compound comprising or consisting of a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 11-64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 11-64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 11-64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide consisting of 10 to 30 linked nucleosides having a nucleobase sequence comprising at least 8, 9, 10, 11, or 12 contiguous nucleobases of any one of SEQ ID NOs: 15, 21, 29, or 64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide consisting of 10 to 30 linked nucleosides having a nucleobase sequence comprising any one of SEQ ID NOs: 15, 21, 29, or 64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ ID NOs: 15, 21, 29, or 64. In certain embodiments, the P23H rhodopsin specific inhibitor is ISIS 564426, ISIS 664844, ISIS 664867, or ISIS 664884. In any of the foregoing embodiments, the antisense compound can be a single-stranded oligonucleotide. In certain embodiments, the antisense compound is administered to the subject by intravitreally such as by intravitreal injection.

In certain embodiments, a method of inhibiting expression of P23H rhodopsin in a cell comprises contacting the cell with a P23H rhodopsin specific inhibitor to the subject. In certain embodiments, the cell is a rod photoreceptor cell or cone cell. In certain embodiments, the cell is in the eye of a subject. In certain embodiments, the cell is in the retina of the eye. In certain embodiments, the inhibitor is an antisense compound targeted to P23H rhodopsin. In certain embodiments, the antisense compound is allele-specific for P23H Rhodospin and selectively inhibits expression of P23H rhodopsin over wild-type rhodopsin. In certain embodiments, the P23H rhodopsin specific inhibitor is a compound comprising or consisting of a modified oligonucleotide consisting of 8 to 80 linked nucleosides complementary within nucleotides 157-174, 157-171, 157-172, or 159-174 of SEQ ID NO: 2. In certain embodiments, the P23H rhodopsin specific inhibitor is a compound comprising or consisting of a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 11-64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 11-64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 11-64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide consisting of 10 to 30 linked nucleosides having a nucleobase sequence comprising at least 8, 9, 10, 11, or 12 contiguous nucleobases of any one of SEQ ID NOs: 15, 21, 29, or 64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide consisting of 10 to 30 linked nucleosides having a nucleobase sequence comprising any one of SEQ ID NOs: 15, 21, 29, or 64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ ID NOs: 15, 21, 29, or 64. In certain embodiments, the P23H rhodopsin specific inhibitor is ISIS 564426, ISIS 664844, ISIS 664867, or ISIS 664884. In any of the foregoing embodiments, the antisense compound can be a single-stranded oligonucleotide.

Certain embodiments are drawn to a P23H rhodopsin specific inhibitor for use in treating retinitis pigmentosa (RP), such as autosomal dominant retinitis pigmentosa (AdRP) associated with P23H rhodopsin. In certain embodiments, the inhibitor is an antisense compound targeted to P23H rhodopsin. In certain embodiments, the antisense compound is allele-specific for P23H Rhodospin and selectively inhibits expression of P23H rhodopsin over wild-type rhodopsin. In certain embodiments, the P23H rhodopsin specific inhibitor is a compound comprising or consisting of a modified oligonucleotide consisting of 8 to 80 linked nucleosides complementary within nucleotides 157-174, 157-171, 157-172, or 159-174 of SEQ ID NO: 2. In certain embodiments, the P23H rhodopsin specific inhibitor is a compound comprising or consisting of a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 11-64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 11-64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 11-64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide consisting of 10 to 30 linked nucleosides having a nucleobase sequence comprising at least 8, 9, 10, 11, or 12 contiguous nucleobases of any one of SEQ ID NOs: 15, 21, 29, or 64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide consisting of 10 to 30 linked nucleosides having a nucleobase sequence comprising any one of SEQ ID NOs: 15, 21, 29, or 64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ ID NOs: 15, 21, 29, or 64. In certain embodiments, the P23H rhodopsin specific inhibitor is ISIS 564426, ISIS 664844, ISIS 664867, or ISIS 664884. In any of the foregoing embodiments, the antisense compound can be a single-stranded oligonucleotide.

Certain embodiments are drawn to a P23H rhodopsin specific inhibitor for use in improving or preserving visual function, visual field, photoreceptor cell function, ERG response, visual acuity, and/or vision-related quality of life of a subject having retinitis pigmentosa (RP), such as autosomal dominant retinitis pigmentosa (AdRP) associated with P23H rhodopsin. Certain embodiments are drawn to a P23H rhodopsin specific inhibitor for use in inhibiting, preventing, or delaying progression of photoreceptor cell loss and/or deterioration of the retina outer nuclear layer (ONL) in a subject having retinitis pigmentosa (RP), such as autosomal dominant retinitis pigmentosa (AdRP) associated with P23H rhodopsin. In certain embodiments, the P23H rhodopsin specific inhibitor is a compound comprising or consisting of a modified oligonucleotide consisting of 8 to 80 linked nucleosides complementary within nucleotides 157-174, 157-171, 157-172, or 159-174 of SEQ ID NO: 2. In certain embodiments, the P23H rhodopsin specific inhibitor is a compound comprising or consisting of a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 11-64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 11-64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 11-64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide consisting of 10 to 30 linked nucleosides having a nucleobase sequence comprising at least 8, 9, 10, 11, or 12 contiguous nucleobases of any one of SEQ ID NOs: 15, 21, 29, or 64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide consisting of 10 to 30 linked nucleosides having a nucleobase sequence comprising any one of SEQ ID NOs: 15, 21, 29, or 64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ ID NOs: 15, 21, 29, or 64. In certain embodiments, the P23H rhodopsin specific inhibitor is ISIS 564426, ISIS 664844, ISIS 664867, or ISIS 664884. In any of the foregoing embodiments, the antisense compound can be a single-stranded oligonucleotide.

Certain embodiments are drawn to use of a P23H rhodopsin specific inhibitor for the manufacture of a medicament for treating retinitis pigmentosa (RP), such as autosomal dominant retinitis pigmentosa (AdRP) associated with P23H rhodopsin. In certain embodiments, the inhibitor is an antisense compound targeted to P23H rhodopsin. In certain embodiments, the antisense compound is allele-specific for P23H Rhodospin and selectively inhibits expression of P23H rhodopsin over wild-type rhodopsin. In certain embodiments, the P23H rhodopsin specific inhibitor is a compound comprising or consisting of a modified oligonucleotide consisting of 8 to 80 linked nucleosides complementary within nucleotides 157-174, 157-171, 157-172, or 159-174 of SEQ ID NO: 2. In certain embodiments, the P23H rhodopsin specific inhibitor is a compound comprising or consisting of a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 11-64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 11-64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 11-64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide consisting of 10 to 30 linked nucleosides having a nucleobase sequence comprising at least 8, 9, 10, 11, or 12 contiguous nucleobases of any one of SEQ ID NOs: 15, 21, 29, or 64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide consisting of 10 to 30 linked nucleosides having a nucleobase sequence comprising any one of SEQ ID NOs: 15, 21, 29, or 64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ ID NOs: 15, 21, 29, or 64. In certain embodiments, the P23H rhodopsin specific inhibitor is ISIS 564426, ISIS 664844, ISIS 664867, or ISIS 664884. In any of the foregoing embodiments, the antisense compound can be a single-stranded oligonucleotide.

Certain embodiments are drawn to use of a P23H rhodopsin specific inhibitor for the manufacture of a medicament for improving or preserving visual function, visual field, photoreceptor cell function, ERG response, visual acuity, and/or vision-related quality of life of a subject having retinitis pigmentosa (RP), such as autosomal dominant retinitis pigmentosa (AdRP) associated with P23H rhodopsin. Certain embodiments are drawn to use of a P23H rhodopsin specific inhibitor for the manufacture of a medicament for inhibiting, preventing, or delaying progression of photoreceptor cell loss and/or deterioration of the retina outer nuclear layer (ONL) in a subject having retinitis pigmentosa (RP), such as autosomal dominant retinitis pigmentosa (AdRP) associated with P23H rhodopsin. In certain embodiments, the inhibitor is an antisense compound targeted to P23H rhodopsin. In certain embodiments, the antisense compound is allele-specific for P23H Rhodospin and selectively inhibits expression of P23H rhodopsin over wild-type rhodopsin. In certain embodiments, the P23H rhodopsin specific inhibitor is a compound comprising or consisting of a modified oligonucleotide consisting of 8 to 80 linked nucleosides complementary within nucleotides 157-174, 157-171, 157-172, or 159-174 of SEQ ID NO: 2. In certain embodiments, the P23H rhodopsin specific inhibitor is a compound comprising or consisting of a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising at least 8 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 11-64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 11-64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide consisting of the nucleobase sequence of any one of SEQ ID NOs: 11-64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide consisting of 10 to 30 linked nucleosides having a nucleobase sequence comprising at least 8, 9, 10, 11, or 12 contiguous nucleobases of any one of SEQ ID NOs: 15, 21, 29, or 64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide consisting of 10 to 30 linked nucleosides having a nucleobase sequence comprising any one of SEQ ID NOs: 15, 21, 29, or 64. In certain embodiments, the P23H rhodopsin specific inhibitor is an antisense compound comprising or consisting of a modified oligonucleotide having a nucleobase sequence consisting of any one of SEQ ID NOs: 15, 21, 29, or 64. In certain embodiments, the P23H rhodopsin specific inhibitor is ISIS 564426, ISIS 664844, ISIS 664867, or ISIS 664884. In any of the foregoing embodiments, the antisense compound can be a single-stranded oligonucleotide.

In any of the foregoing methods or uses, the P23H rhodopsin specific inhibitor can be an antisense compound targeted to P23H rhodopsin. In certain embodiments, the antisense compound is an antisense oligonucleotide, for example an antisense oligonucleotide consisting of 8 to 80 linked nucleosides, 12 to 30 linked nucleosides, or 20 linked nucleosides. In certain embodiments, the antisense oligonucleotide is at least 80%, 85%, 90%, 95% or 100% complementary to any of the nucleobase sequences recited in SEQ ID NOs: 1-4. In certain embodiments, the antisense oligonucleotide comprises at least one modified internucleoside linkage, at least one modified sugar and/or at least one modified nucleobase. In certain embodiments, the modified internucleoside linkage is a phosphorothioate internucleoside linkage, the modified sugar is a bicyclic sugar or a 2′-O-methoxyethyl, and the modified nucleobase is a 5-methylcytosine. In certain embodiments, the modified oligonucleotide comprises a gap segment consisting of linked deoxynucleosides; a 5′ wing segment consisting of linked nucleosides; and a 3′ wing segment consisting of linked nucleosides, wherein the gap segment is positioned immediately adjacent to and between the 5′ wing segment and the 3′ wing segment and wherein each nucleoside of each wing segment comprises a modified sugar. In certain embodiments, the antisense oligonucleotide is allele-specific for P23H Rhodospin and selectively inhibits expression of P23H rhodopsin over wild-type rhodopsin.

In any of the foregoing methods or uses, the P23H rhodopsin specific inhibitor can be a compound comprising or consisting of a modified oligonucleotide consisting of 10 to 30 linked nucleosides having a nucleobase sequence comprising any one of SEQ ID NOs: 11-64, wherein the modified oligonucleotide comprises:

-   -   a gap segment consisting of linked deoxynucleosides;     -   a 5′ wing segment consisting of linked nucleosides; and     -   a 3′ wing segment consisting of linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.

In any of the foregoing methods or uses, the P23H rhodopsin specific inhibitor can be a compound comprising or consisting of a modified oligonucleotide consisting of 10 to 30 linked nucleosides having a nucleobase sequence comprising any one of SEQ ID NOs: 15, 21, 29, or 64, wherein the modified oligonucleotide comprises:

-   -   a gap segment consisting of linked deoxynucleosides;     -   a 5′ wing segment consisting of linked nucleosides; and     -   a 3′ wing segment consisting of linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.

In any of the foregoing methods or uses, the P23H rhodopsin specific inhibitor can be a compound comprising or consisting of a modified oligonucleotide consisting of 16 to 30 linked nucleosides having a nucleobase sequence comprising the sequence recited in SEQ ID NO: 15, wherein the modified oligonucleotide comprises:

-   -   a gap segment consisting of ten linked deoxynucleosides;     -   a 5′ wing segment consisting of three linked nucleosides; and     -   a 3′ wing segment consisting of three linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment; wherein each nucleoside of each wing segment comprises a cEt sugar; wherein each internucleoside linkage is a phosphorothioate linkage; and wherein each cytosine is a 5-methylcytosine.

In any of the foregoing methods or uses, the P23H rhodopsin specific inhibitor can be a compound comprising or consisting of a modified oligonucleotide consisting of 16 linked nucleosides having a nucleobase sequence consisting of the sequence recited in SEQ ID NO: 15, wherein the modified oligonucleotide comprises:

-   -   a gap segment consisting of ten linked deoxynucleosides;     -   a 5′ wing segment consisting of three linked nucleosides; and     -   a 3′ wing segment consisting of three linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment; wherein each nucleoside of each wing segment comprises a cEt sugar; wherein each internucleoside linkage is a phosphorothioate linkage; and wherein each cytosine is a 5-methylcytosine.

In any of the foregoing methods or uses, the P23H rhodopsin specific inhibitor can be a compound comprising or consisting of a modified oligonucleotide consisting of 16 to 30 linked nucleosides having a nucleobase sequence comprising the sequence recited in SEQ ID NO: 64, wherein the modified oligonucleotide comprises:

-   -   a gap segment consisting of nine linked deoxynucleosides;     -   a 5′ wing segment consisting of four linked nucleosides; and     -   a 3′ wing segment consisting of three linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment; wherein the 5′ wing segment comprises a cEt sugar, a cEt sugar, a cEt sugar, and a 2′-flouro sugar in the 5′ to 3′ direction; wherein each nucleoside of the 3′ wing segment comprises a cEt sugar; wherein each internucleoside linkage is a phosphorothioate linkage; and wherein each cytosine is a 5-methylcytosine.

In any of the foregoing methods or uses, the P23H rhodopsin specific inhibitor can be a compound comprising or consisting of a modified oligonucleotide consisting of 16 linked nucleosides having a nucleobase sequence consisting of the sequence recited in SEQ ID NO: 64, wherein the modified oligonucleotide comprises:

-   -   a gap segment consisting of nine linked deoxynucleosides;     -   a 5′ wing segment consisting of four linked nucleosides; and     -   a 3′ wing segment consisting of three linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment; wherein the 5′ wing segment comprises a cEt sugar, a cEt sugar, a cEt sugar, and a 2′-flouro sugar in the 5′ to 3′ direction; wherein each nucleoside of the 3′ wing segment comprises a cEt sugar; wherein each internucleoside linkage is a phosphorothioate linkage; and wherein each cytosine is a 5-methylcytosine.

In any of the foregoing methods or uses, the P23H rhodopsin specific inhibitor can be a compound comprising or consisting of a modified oligonucleotide consisting of 16 to 30 linked nucleosides having a nucleobase sequence comprising the sequence recited in SEQ ID NO: 21, wherein the modified oligonucleotide comprises:

-   -   a gap segment consisting of ten linked deoxynucleosides;     -   a 5′ wing segment consisting of two linked nucleosides; and     -   a 3′ wing segment consisting of four linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment; wherein each nucleoside of the 5′ wing segment comprises a cEt sugar; wherein the 3′ wing segment comprises a cEt sugar, a 2′-O-methoxyethyl sugar, a cEt sugar, and a 2′-O-methoxyethyl sugar in the 5′ to 3′ direction; wherein each internucleoside linkage is a phosphorothioate linkage; and wherein each cytosine is a 5-methylcytosine.

In any of the foregoing methods or uses, the P23H rhodopsin specific inhibitor can be a compound comprising or consisting of a modified oligonucleotide consisting of 16 linked nucleosides having a nucleobase sequence consisting of the sequence recited in SEQ ID NO: 21, wherein the modified oligonucleotide comprises:

-   -   a gap segment consisting often linked deoxynucleosides;     -   a 5′ wing segment consisting of two linked nucleosides; and     -   a 3′ wing segment consisting of four linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment; wherein each nucleoside of the 5′ wing segment comprises a cEt sugar; wherein the 3′ wing segment comprises a cEt sugar, a 2′-O-methoxyethyl sugar, a cEt sugar, and a 2′-O-methoxyethyl sugar in the 5′ to 3′ direction; wherein each internucleoside linkage is a phosphorothioate linkage; and wherein each cytosine is a 5-methylcytosine.

In any of the foregoing methods or uses, the P23H rhodopsin specific inhibitor can be a compound comprising or consisting of a modified oligonucleotide consisting of 15 to 30 linked nucleosides having a nucleobase sequence comprising the sequence recited in SEQ ID NO: 29, wherein the modified oligonucleotide comprises:

-   -   a gap segment consisting of ten linked deoxynucleosides;     -   a 5′ wing segment consisting of two linked nucleosides; and     -   a 3′ wing segment consisting of three linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment; wherein each nucleoside of the 5′ wing segment comprises a cEt sugar; wherein the 3′ wing segment comprises a cEt sugar, a 2′-O-methoxyethyl sugar, and a cEt sugar in the 5′ to 3′ direction; wherein each internucleoside linkage is a phosphorothioate linkage; and wherein each cytosine is a 5-methylcytosine.

In any of the foregoing methods or uses, the P23H rhodopsin specific inhibitor can be a compound comprising or consisting of a modified oligonucleotide consisting of 15 linked nucleosides having a nucleobase sequence consisting of the sequence recited in SEQ ID NO: 29, wherein the modified oligonucleotide comprises:

-   -   a gap segment consisting of ten linked deoxynucleosides;     -   a 5′ wing segment consisting of two linked nucleosides; and     -   a 3′ wing segment consisting of three linked nucleosides;

wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment; wherein each nucleoside of the 5′ wing segment comprises a cEt sugar; wherein the 3′ wing segment comprises a cEt sugar, a 2′-O-methoxyethyl sugar, and a cEt sugar in the 5′ to 3′ direction; wherein each internucleoside linkage is a phosphorothioate linkage; and wherein each cytosine is a 5-methylcytosine.

In any of the foregoing methods or uses, the P23H rhodopsin specific inhibitor is administered intravitreally, such as by intravitreal injection.

Antisense Compounds

Oligomeric compounds include, but are not limited to, oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, antisense compounds, antisense oligonucleotides, and siRNAs. An oligomeric compound may be “antisense” to a target nucleic acid, meaning that is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.

In certain embodiments, an antisense compound has a nucleobase sequence that, when written in the 5′ to 3′ direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted.

In certain embodiments, an antisense compound is 10 to 30 subunits in length. In certain embodiments, an antisense compound is 12 to 30 subunits in length. In certain embodiments, an antisense compound is 12 to 22 subunits in length. In certain embodiments, an antisense compound is 14 to 30 subunits in length. In certain embodiments, an antisense compound is 14 to 20 subunits in length. In certain embodiments, an antisense compound is 15 to 30 subunits in length. In certain embodiments, an antisense compound is 15 to 20 subunits in length. In certain embodiments, an antisense compound is 16 to 30 subunits in length. In certain embodiments, an antisense compound is 16 to 20 subunits in length. In certain embodiments, an antisense compound is 17 to 30 subunits in length. In certain embodiments, an antisense compound is 17 to 20 subunits in length. In certain embodiments, an antisense compound is 18 to 30 subunits in length. In certain embodiments, an antisense compound is 18 to 21 subunits in length. In certain embodiments, an antisense compound is 18 to 20 subunits in length. In certain embodiments, an antisense compound is 20 to 30 subunits in length. In other words, such antisense compounds are from 12 to 30 linked subunits, 14 to 30 linked subunits, 14 to 20 subunits, 15 to 30 subunits, 15 to 20 subunits, 16 to 30 subunits, 16 to 20 subunits, 17 to 30 subunits, 17 to 20 subunits, 18 to 30 subunits, 18 to 20 subunits, 18 to 21 subunits, 20 to 30 subunits, or 12 to 22 linked subunits, respectively. In certain embodiments, an antisense compound is 14 subunits in length. In certain embodiments, an antisense compound is 16 subunits in length. In certain embodiments, an antisense compound is 17 subunits in length. In certain embodiments, an antisense compound is 18 subunits in length. In certain embodiments, an antisense compound is 19 subunits in length. In certain embodiments, an antisense compound is 20 subunits in length. In other embodiments, the antisense compound is 8 to 80, 12 to 50, 13 to 30, 13 to 50, 14 to 30, 14 to 50, 15 to 30, 15 to 50, 16 to 30, 16 to 50, 17 to 30, 17 to 50, 18 to 22, 18 to 24, 18 to 30, 18 to 50, 19 to 22, 19 to 30, 19 to 50, or 20 to 30 linked subunits. In certain such embodiments, the antisense compounds are 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linked subunits in length, or a range defined by any two of the above values. In some embodiments the antisense compound is an antisense oligonucleotide, and the linked subunits are nucleotides.

In certain embodiments antisense oligonucleotides may be shortened or truncated. For example, a single subunit may be deleted from the 5′ end (5′ truncation), or alternatively from the 3′ end (3′ truncation). A shortened or truncated antisense compound targeted to an P23H rhodopsin nucleic acid may have two subunits deleted from the 5′ end, or alternatively may have two subunits deleted from the 3′ end, of the antisense compound. Alternatively, the deleted nucleosides may be dispersed throughout the antisense compound, for example, in an antisense compound having one nucleoside deleted from the 5′ end and one nucleoside deleted from the 3′ end.

When a single additional subunit is present in a lengthened antisense compound, the additional subunit may be located at the 5′ or 3′ end of the antisense compound. When two or more additional subunits are present, the added subunits may be adjacent to each other, for example, in an antisense compound having two subunits added to the 5′ end (5′ addition), or alternatively to the 3′ end (3′ addition), of the antisense compound. Alternatively, the added subunits may be dispersed throughout the antisense compound, for example, in an antisense compound having one subunit added to the 5′ end and one subunit added to the 3′ end.

It is possible to increase or decrease the length of an antisense compound, such as an antisense oligonucleotide, and/or introduce mismatch bases without eliminating activity. For example, in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series of antisense oligonucleotides 13-25 nucleobases in length were tested for their ability to induce cleavage of a target RNA in an oocyte injection model. Antisense oligonucleotides 25 nucleobases in length with 8 or 11 mismatch bases near the ends of the antisense oligonucleotides were able to direct specific cleavage of the target mRNA, albeit to a lesser extent than the antisense oligonucleotides that contained no mismatches. Similarly, target specific cleavage was achieved using 13 nucleobase antisense oligonucleotides, including those with 1 or 3 mismatches.

Gautschi el al. (J Natl. Cancer Inst. 93:463-471, March 2001) demonstrated the ability of an oligonucleotide having 100% complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xL mRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and in vivo. Furthermore, this oligonucleotide demonstrated potent anti-tumor activity in vivo.

Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested a series of tandem 14 nucleobase antisense oligonucleotides, and a 28 and 42 nucleobase antisense oligonucleotides comprised of the sequence of two or three of the tandem antisense oligonucleotides, respectively, for their ability to arrest translation of human DHFR in a rabbit reticulocyte assay. Each of the three 14 nucleobase antisense oligonucleotides alone was able to inhibit translation, albeit at a more modest level than the 28 or 42 nucleobase antisense oligonucleotides.

Certain Antisense Compound Motifs and Mechanisms

In certain embodiments, antisense compounds have chemically modified subunits arranged in patterns, or motifs, to confer to the antisense compounds properties such as enhanced inhibitory activity, increased binding affinity for a target nucleic acid, or resistance to degradation by in vivo nucleases.

Chimeric antisense compounds typically contain at least one region modified so as to confer increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity for the target nucleic acid, and/or increased inhibitory activity. A second region of a chimeric antisense compound may confer another desired property e.g., serve as a substrate for the cellular endonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex.

Antisense activity may result from any mechanism involving the hybridization of the antisense compound (e.g., oligonucleotide) with a target nucleic acid, wherein the hybridization ultimately results in a biological effect. In certain embodiments, the amount and/or activity of the target nucleic acid is modulated. In certain embodiments, the amount and/or activity of the target nucleic acid is reduced. In certain embodiments, hybridization of the antisense compound to the target nucleic acid ultimately results in target nucleic acid degradation. In certain embodiments, hybridization of the antisense compound to the target nucleic acid does not result in target nucleic acid degradation. In certain such embodiments, the presence of the antisense compound hybridized with the target nucleic acid (occupancy) results in a modulation of antisense activity. In certain embodiments, antisense compounds having a particular chemical motif or pattern of chemical modifications are particularly suited to exploit one or more mechanisms. In certain embodiments, antisense compounds function through more than one mechanism and/or through mechanisms that have not been elucidated. Accordingly, the antisense compounds described herein are not limited by particular mechanism.

Antisense mechanisms include, without limitation, RNase H mediated antisense; RNAi mechanisms, which utilize the RISC pathway and include, without limitation, siRNA, ssRNA and microRNA mechanisms; and occupancy based mechanisms. Certain antisense compounds may act through more than one such mechanism and/or through additional mechanisms.

RNase H-Mediated Antisense

In certain embodiments, antisense activity results at least in part from degradation of target RNA by RNase H. RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds which are “DNA-like” elicit RNase H activity in mammalian cells. Accordingly, antisense compounds comprising at least a portion of DNA or DNA-like nucleosides may activate RNase H, resulting in cleavage of the target nucleic acid. In certain embodiments, antisense compounds that utilize RNase H comprise one or more modified nucleosides. In certain embodiments, such antisense compounds comprise at least one block of 1-8 modified nucleosides. In certain such embodiments, the modified nucleosides do not support RNase H activity. In certain embodiments, such antisense compounds are gapmers, as described herein. In certain such embodiments, the gap of the gapmer comprises DNA nucleosides. In certain such embodiments, the gap of the gapmer comprises DNA-like nucleosides. In certain such embodiments, the gap of the gapmer comprises DNA nucleosides and DNA-like nucleosides.

Certain antisense compounds having a gapmer motif are considered chimeric antisense compounds. In a gapmer an internal region having a plurality of nucleotides that supports RNaseH cleavage is positioned between external regions having a plurality of nucleotides that are chemically distinct from the nucleosides of the internal region. In the case of an antisense oligonucleotide having a gapmer motif, the gap segment generally serves as the substrate for endonuclease cleavage, while the wing segments comprise modified nucleosides. In certain embodiments, the regions of a gapmer are differentiated by the types of sugar moieties comprising each distinct region. The types of sugar moieties that are used to differentiate the regions of a gapmer may in some embodiments include β-D-ribonucleosides, β-D-deoxyribonucleosides, 2′-modified nucleosides (such 2′-modified nucleosides may include 2′-MOE and 2′-O—CH₃, among others), and bicyclic sugar modified nucleosides (such bicyclic sugar modified nucleosides may include those having a constrained ethyl). In certain embodiments, nucleosides in the wings may include several modified sugar moieties, including, for example 2′-MOE and bicyclic sugar moieties such as constrained ethyl or LNA. In certain embodiments, wings may include several modified and unmodified sugar moieties. In certain embodiments, wings may include various combinations of 2′-MOE nucleosides, bicyclic sugar moieties such as constrained ethyl nucleosides or LNA nucleosides, and 2′-deoxynucleosides.

Each distinct region may comprise uniform sugar moieties, variant, or alternating sugar moieties. The wing-gap-wing motif is frequently described as “X—Y—Z”, where “X” represents the length of the 5′-wing, “Y” represents the length of the gap, and “Z” represents the length of the 3′-wing. “X” and “Z” may comprise uniform, variant, or alternating sugar moieties. In certain embodiments, “X” and “Y” may include one or more 2′-deoxynucleosides. “Y” may comprise 2′-deoxynucleosides. As used herein, a gapmer described as “X—Y—Z” has a configuration such that the gap is positioned immediately adjacent to each of the 5′-wing and the 3′ wing. Thus, no intervening nucleotides exist between the 5′-wing and gap, or the gap and the 3′-wing. Any of the antisense compounds described herein can have a gapmer motif. In certain embodiments, “X” and “Z” are the same; in other embodiments they are different. In certain embodiments, “Y” is between 8 and 15 nucleosides. X, Y, or Z can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more nucleosides.

In certain embodiments, the antisense compound targeted to a P23H rhodopsin nucleic acid has a gapmer motif in which the gap consists of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 linked nucleosides.

In certain embodiments, the antisense oligonucleotide has a sugar motif described by Formula A as follows: (J)_(m)-(B)_(n)-(J)_(p)-(B)_(r)-(A)_(t)-(D)_(g)-(A)_(v)-(B)_(w)-(J)_(x)-(B)_(y)-(J)_(z)

wherein:

each A is independently a 2′-substituted nucleoside;

each B is independently a bicyclic nucleoside;

each J is independently either a 2′-substituted nucleoside or a 2′-deoxynucleoside;

each D is a 2′-deoxynucleoside;

m is 0-4; n is 0-2; p is 0-2; r is 0-2; t is 0-2; v is 0-2; w is 0-4; x is 0-2; y is 0-2; z is 0-4; g is 6-14; provided that:

at least one of m, n, and r is other than 0;

at least one of w and y is other than 0;

the sum of m, n, p, r, and t is from 2 to 5; and

the sum of v, w, x, y, and z is from 2 to 5.

RNAi Compounds

In certain embodiments, antisense compounds are interfering RNA compounds (RNAi), which include double-stranded RNA compounds (also referred to as short-interfering RNA or siRNA) and single-stranded RNAi compounds (or ssRNA). Such compounds work at least in part through the RISC pathway to degrade and/or sequester a target nucleic acid (thus, include microRNA/microRNA-mimic compounds). In certain embodiments, antisense compounds comprise modifications that make them particularly suited for such mechanisms.

i. ssRNA Compounds

In certain embodiments, antisense compounds including those particularly suited for use as single-stranded RNAi compounds (ssRNA) comprise a modified 5′-terminal end. In certain such embodiments, the 5′-terminal end comprises a modified phosphate moiety. In certain embodiments, such modified phosphate is stabilized (e.g., resistant to degradation/cleavage compared to unmodified 5′-phosphate). In certain embodiments, such 5′-terminal nucleosides stabilize the 5′-phosphorous moiety. Certain modified 5′-terminal nucleosides may be found in the art, for example in WO/2011/139702.

In certain embodiments the 5′-nucleoside of an ssRNA compound has Formula IIc:

wherein:

T₁ is an optionally protected phosphorus moiety;

T₂ is an internucleoside linking group linking the compound of Formula IIe to the oligomeric compound;

A has one of the formulas:

Q₁ and Q₂ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl or N(R₃)(R₄);

Q₃ is O, S, N(R₅) or C(R₆)(R₇);

each R₃, R₄, R₅, R₆ and R₇ is, independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl or C₁-C₆ alkoxy;

M₃ is O, S, NR₁₄, C(R₁₅)(R₁₆), C(R₁₅)(R₁₆)C(R₁₇)(R₁₈), C(R₁₅)═C(R₁₇), OC(R₁₅)(R₁₆) or OC(R₁₅)(Bx₂);

R₁₄ is H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

R₁₅, R₁₆, R₁₇ and R₁₈ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

Bx₁ is a heterocyclic base moiety;

or if Bx₂ is present then Bx₂ is a heterocyclic base moiety and Bx₁ is H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

J₄, J₅, J₆ and J₇ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

or J₄ forms a bridge with one of J₅ or J₇ wherein said bridge comprises from 1 to 3 linked biradical groups selected from O, S, NR₁₉, C(R₂₀)(R₂₁), C(R₂₀)═C(R₂₁), C[═C(R₂₀)(R₂₁)] and C(═O) and the other two of J₅, J₆ and J₇ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

each R₁₉, R₂₀ and R₂₁ is, independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

G is H, OH, halogen or O—[C(R₈)(R₉)]_(n)[(C═O)_(m)—X₁]_(j)—Z;

each R₈ and R₉ is, independently, H, halogen, C₁-C₆ alkyl or substituted C₁-C₆ alkyl;

X₁ is O, S or N(E₁);

Z is H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl or N(E₂)(E₃);

E₁, E₂ and E₃ are each, independently, H, C₁-C₆ alkyl or substituted C₁-C₆ alkyl;

n is from 1 to about 6;

m is 0 or 1;

j is 0 or 1;

each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJ₁, N(J₁)(J₂), =NJ₁, SJ₁, N₃, CN, OC(═X₂)J₁, OC(═X₂)N(J₁)(J₂) and C(═X₂)N(J₁)(J₂);

X₂ is O, S or NJ₃;

each J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl;

when j is 1 then Z is other than halogen or N(E₂)(E₃); and

wherein said oligomeric compound comprises from 8 to 40 monomeric subunits and is hybridizable to at least a portion of a target nucleic acid.

In certain embodiments, M₃ is O, CH═CH, OCH₂ or OC(H)(Bx₂). In certain embodiments, M₃ is O.

In certain embodiments, J₄, J₅, J₆ and J₇ are each H. In certain embodiments, J₄ forms a bridge with one of J₅ or J₇.

In certain embodiments, A has one of the formulas:

wherein:

Q₁ and Q₂ are each, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy or substituted C₁-C₆ alkoxy. In certain embodiments, Q₁ and Q₂ are each H. In certain embodiments, Q₁ and Q₂ are each, independently, H or halogen. In certain embodiments, Q₁ and Q₂ is H and the other of Q₁ and Q₂ is F, CH₃ or OCH₃.

In certain embodiments, T₁ has the formula:

wherein:

R_(a) and R_(c) are each, independently, protected hydroxyl, protected thiol, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₁-C₆ alkoxy, substituted C₁-C₆ alkoxy, protected amino or substituted amino; and

R_(b) is O or S. In certain embodiments, R_(b) is O and R_(a) and R_(c) are each, independently, OCH₃, OCH₂CH₃ or CH(CH₃)₂.

In certain embodiments, G is halogen, OCH₃, OCH₂F, OCHF₂, OCF₃, OCH₂CH₃, O(CH₂)₂F, OCH₂CHF₂, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃, O(CH₂)₂—SCH₃, O(CH₂)₂—OCF₃, O(CH₂)₃—N(R₁₀)(R₁₁), O(CH₂)₂—ON(R₁₀)(R₁₁), O(CH₂)₂—O(CH₂)₂—N(R₁₀)(R₁₁), OCH₂C(═O)—N(R₁₀)(R₁), OCH₂C(═O)—N(R₁₂)—(CH₂)₂—N(R₁₀)(R₁₁) or O(CH₂)₂—N(R₁₂)—C(═NR₁₃)[N(R₁₀)(R₁₁)] wherein R₁₀, R₁₁, R₁₂ and R₁₃ are each, independently, H or C₁-C₆ alkyl. In certain embodiments, G is halogen, OCH₃, OCF₃, OCH₂CH₃, OCH₂CF₃, OCH₂—CH═CH₂, O(CH₂)₂—OCH₃, O(CH₂)₂—O(CH₂)₂—N(CH₃)₂, OCH₂C(═O)—N(H)CH₃, OCH₂C(═O)—N(H)—(CH₂)₂—N(CH₃)₂ or OCH₂—N(H)—C(═NH)NH₂. In certain embodiments, G is F, OCH₃ or O(CH₂)₂—OCH₃. In certain embodiments, G is O(CH₂)₂—OCH₃.

In certain embodiments, the 5′-terminal nucleoside has Formula IIe:

In certain embodiments, antisense compounds, including those particularly suitable for ssRNA comprise one or more type of modified sugar moieties and/or naturally occurring sugar moieties arranged along an oligonucleotide or region thereof in a defined pattern or sugar modification motif. Such motifs may include any of the sugar modifications discussed herein and/or other known sugar modifications.

In certain embodiments, the oligonucleotides comprise or consist of a region having uniform sugar modifications. In certain such embodiments, each nucleoside of the region comprises the same RNA-like sugar modification. In certain embodiments, each nucleoside of the region is a 2′-F nucleoside. In certain embodiments, each nucleoside of the region is a 2′-OMe nucleoside. In certain embodiments, each nucleoside of the region is a 2′-MOE nucleoside. In certain embodiments, each nucleoside of the region is a cEt nucleoside. In certain embodiments, each nucleoside of the region is an LNA nucleoside. In certain embodiments, the uniform region constitutes all or essentially all of the oligonucleotide. In certain embodiments, the region constitutes the entire oligonucleotide except for 1-4 terminal nucleosides.

In certain embodiments, oligonucleotides comprise one or more regions of alternating sugar modifications, wherein the nucleosides alternate between nucleotides having a sugar modification of a first type and nucleotides having a sugar modification of a second type. In certain embodiments, nucleosides of both types are RNA-like nucleosides. In certain embodiments the alternating nucleosides are selected from: 2′-OMe, 2′-F, 2′-MOE, LNA, and cEt. In certain embodiments, the alternating modifications are 2′-F and 2′-OMe. Such regions may be contiguous or may be interrupted by differently modified nucleosides or conjugated nucleosides.

In certain embodiments, the alternating region of alternating modifications each consist of a single nucleoside (i.e., the pattern is (AB)_(x)A_(y) wherein A is a nucleoside having a sugar modification of a first type and B is a nucleoside having a sugar modification of a second type; x is 1-20 and y is 0 or 1). In certain embodiments, one or more alternating regions in an alternating motif includes more than a single nucleoside of a type. For example, oligonucleotides may include one or more regions of any of the following nucleoside motifs:

AABBAA;

ABBABB;

AABAAB;

ABBABAABB;

ABABAA;

AABABAB;

ABABAA;

ABBAABBABABAA;

BABBAABBABABAA; or

ABABBAABBABABAA;

wherein A is a nucleoside of a first type and B is a nucleoside of a second type. In certain embodiments, A and B are each selected from 2′-F, 2′-OMe, BNA, and MOE.

In certain embodiments, oligonucleotides having such an alternating motif also comprise a modified 5′ terminal nucleoside, such as those of formula IIe or IIe.

In certain embodiments, oligonucleotides comprise a region having a 2-2-3 motif Such regions comprises the following motif: -(A)₂-(B)_(x)-(A)₂-(C)_(y)-(A)₃- wherein: A is a first type of modified nucleoside;

B and C, are nucleosides that are differently modified than A, however, B and C may have the same or different modifications as one another;

x and y are from 1 to 15.

In certain embodiments, A is a 2′-OMe modified nucleoside. In certain embodiments, B and C are both 2′-F modified nucleosides. In certain embodiments, A is a 2′-OMe modified nucleoside and B and C are both 2′-F modified nucleosides.

In certain embodiments, oligonucleosides have the following sugar motif: 5′-(Q)-(AB)_(x)A_(y)-(D)_(z) wherein:

Q is a nucleoside comprising a stabilized phosphate moiety. In certain embodiments, Q is a nucleoside having Formula IIc or IIe;

A is a first type of modified nucleoside;

B is a second type of modified nucleoside;

D is a modified nucleoside comprising a modification different from the nucleoside adjacent to it. Thus, if y is 0, then D must be differently modified than B and if y is 1, then D must be differently modified than A. In certain embodiments, D differs from both A and B.

X is 5-15;

Y is 0 or 1;

Z is 0-4.

In certain embodiments, oligonucleosides have the following sugar motif: 5′-(Q)-(A)_(x)-(D)_(z) wherein:

Q is a nucleoside comprising a stabilized phosphate moiety. In certain embodiments, Q is a nucleoside having Formula IIc or IIe;

A is a first type of modified nucleoside; D is a modified nucleoside comprising a modification different from A.

X is 11-30;

Z is 0-4.

In certain embodiments A, B, C, and D in the above motifs are selected from: 2′-OMe, 2′-F, 2′-MOE, LNA, and cEt. In certain embodiments, D represents terminal nucleosides. In certain embodiments, such terminal nucleosides are not designed to hybridize to the target nucleic acid (though one or more might hybridize by chance). In certain embodiments, the nucleobase of each D nucleoside is adenine, regardless of the identity of the nucleobase at the corresponding position of the target nucleic acid. In certain embodiments the nucleobase of each D nucleoside is thymine.

In certain embodiments, antisense compounds, including those particularly suited for use as ssRNA comprise modified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or modified internucleoside linkage motif. In certain embodiments, oligonucleotides comprise a region having an alternating internucleoside linkage motif. In certain embodiments, oligonucleotides comprise a region of uniformly modified internucleoside linkages. In certain such embodiments, the oligonucleotide comprises a region that is uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide is uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate and at least one internucleoside linkage is phosphorothioate.

In certain embodiments, the oligonucleotide comprises at least 6 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 8 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 10 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 6 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 8 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 10 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least one 12 consecutive phosphorothioate internucleoside linkages. In certain such embodiments, at least one such block is located at the 3′ end of the oligonucleotide. In certain such embodiments, at least one such block is located within 3 nucleosides of the 3′ end of the oligonucleotide.

Oligonucleotides having any of the various sugar motifs described herein, may have any linkage motif. For example, the oligonucleotides, including but not limited to those described above, may have a linkage motif selected from non-limiting the table below:

5′ most linkage Central region 3′-region PS Alternating PO/PS 6 PS PS Alternating PO/PS 7 PS PS Alternating PO/PS 8 PS

ii. siRNA Compounds

In certain embodiments, antisense compounds are double-stranded RNAi compounds (siRNA). In such embodiments, one or both strands may comprise any modification motif described above for ssRNA. In certain embodiments, ssRNA compounds may be unmodified RNA. In certain embodiments, siRNA compounds may comprise unmodified RNA nucleosides, but modified internucleoside linkages.

Several embodiments relate to double-stranded compositions wherein each strand comprises a motif defined by the location of one or more modified or unmodified nucleosides. In certain embodiments, compositions are provided comprising a first and a second oligomeric compound that are fully or at least partially hybridized to form a duplex region and further comprising a region that is complementary to and hybridizes to a nucleic acid target. It is suitable that such a composition comprise a first oligomeric compound that is an antisense strand having full or partial complementarity to a nucleic acid target and a second oligomeric compound that is a sense strand having one or more regions of complementarity to and forming at least one duplex region with the first oligomeric compound.

The compositions of several embodiments modulate gene expression by hybridizing to a nucleic acid target resulting in loss of its normal function. In certain embodiments, the degradation of the targeted P23H rhodopsin is facilitated by an activated RISC complex that is formed with compositions of the invention.

Several embodiments are directed to double-stranded compositions wherein one of the strands is useful in, for example, influencing the preferential loading of the opposite strand into the RISC (or cleavage) complex. The compositions are useful for targeting selected nucleic acid molecules and modulating the expression of one or more genes. In some embodiments, the compositions of the present invention hybridize to a portion of a target RNA resulting in loss of normal function of the target RNA.

Certain embodiments are drawn to double-stranded compositions wherein both the strands comprises a hemimer motif, a fully modified motif, a positionally modified motif or an alternating motif. Each strand of the compositions of the present invention can be modified to fulfill a particular role in for example the siRNA pathway. Using a different motif in each strand or the same motif with different chemical modifications in each strand permits targeting the antisense strand for the RISC complex while inhibiting the incorporation of the sense strand. Within this model, each strand can be independently modified such that it is enhanced for its particular role. The antisense strand can be modified at the 5′-end to enhance its role in one region of the RISC while the 3′-end can be modified differentially to enhance its role in a different region of the RISC.

The double-stranded oligonucleotide molecules can be a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The double-stranded oligonucleotide molecules can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary (i.e. each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double-stranded structure, for example wherein the double-stranded region is about 15 to about 30, e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs; the antisense strand comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense strand comprises nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof (e.g., about 15 to about 25 or more nucleotides of the double-stranded oligonucleotide molecule are complementary to the target nucleic acid or a portion thereof). Alternatively, the double-stranded oligonucleotide is assembled from a single oligonucleotide, where the self-complementary sense and antisense regions of the siRNA are linked by means of a nucleic acid based or non-nucleic acid-based linker(s).

The double-stranded oligonucleotide can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The double-stranded oligonucleotide can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating RNAi.

In certain embodiments, the double-stranded oligonucleotide comprises separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linkers molecules as is known in the art, or are alternately non-covalently linked by ionic interactions, hydrogen bonding, van der waals interactions, hydrophobic interactions, and/or stacking interactions. In certain embodiments, the double-stranded oligonucleotide comprises nucleotide sequence that is complementary to nucleotide sequence of a target gene. In another embodiment, the double-stranded oligonucleotide interacts with nucleotide sequence of a target gene in a manner that causes inhibition of expression of the target gene.

As used herein, double-stranded oligonucleotides need not be limited to those molecules containing only RNA, but further encompasses chemically modified nucleotides and non-nucleotides. In certain embodiments, the short interfering nucleic acid molecules lack 2′-hydroxy (2′-OH) containing nucleotides. In certain embodiments short interfering nucleic acids optionally do not include any ribonucleotides (e.g., nucleotides having a 2′—OH group). Such double-stranded oligonucleotides that do not require the presence of ribonucleotides within the molecule to support RNAi can however have an attached linker or linkers or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2′—OH groups. Optionally, double-stranded oligonucleotides can comprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions. As used herein, the term siRNA is meant to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi, for example short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), and others. In addition, as used herein, the term RNAi is meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, translational inhibition, or epigenetics. For example, double-stranded oligonucleotides can be used to epigenetically silence genes at both the post-transcriptional level and the pre-transcriptional level. In a non-limiting example, epigenetic regulation of gene expression by siRNA molecules of the invention can result from siRNA mediated modification of chromatin structure or methylation pattern to alter gene expression (see, for example, Verdel et al., 2004, Science, 303, 672-676; Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237).

It is contemplated that compounds and compositions of several embodiments provided herein can target P23H rhodopsin by a dsRNA-mediated gene silencing or RNAi mechanism, including, e.g., “hairpin” or stem-loop double-stranded RNA effector molecules in which a single RNA strand with self-complementary sequences is capable of assuming a double-stranded conformation, or duplex dsRNA effector molecules comprising two separate strands of RNA. In various embodiments, the dsRNA consists entirely of ribonucleotides or consists of a mixture of ribonucleotides and deoxynucleotides, such as the RNA/DNA hybrids disclosed, for example, by WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999. The dsRNA or dsRNA effector molecule may be a single molecule with a region of self-complementarity such that nucleotides in one segment of the molecule base pair with nucleotides in another segment of the molecule. In various embodiments, a dsRNA that consists of a single molecule consists entirely of ribonucleotides or includes a region of ribonucleotides that is complementary to a region of deoxyribonucleotides. Alternatively, the dsRNA may include two different strands that have a region of complementarity to each other.

In various embodiments, both strands consist entirely of ribonucleotides, one strand consists entirely of ribonucleotides and one strand consists entirely of deoxyribonucleotides, or one or both strands contain a mixture of ribonucleotides and deoxyribonucleotides. In certain embodiments, the regions of complementarity are at least 70, 80, 90, 95, 98, or 100% complementary to each other and to a target nucleic acid sequence. In certain embodiments, the region of the dsRNA that is present in a double-stranded conformation includes at least 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 75, 100, 200, 500, 1000, 2000 or 5000 nucleotides or includes all of the nucleotides in a cDNA or other target nucleic acid sequence being represented in the dsRNA. In some embodiments, the dsRNA does not contain any single stranded regions, such as single stranded ends, or the dsRNA is a hairpin. In other embodiments, the dsRNA has one or more single stranded regions or overhangs. In certain embodiments, RNA/DNA hybrids include a DNA strand or region that is an antisense strand or region (e.g, has at least 70, 80, 90, 95, 98, or 100% complementarity to a target nucleic acid) and an RNA strand or region that is a sense strand or region (e.g, has at least 70, 80, 90, 95, 98, or 100% identity to a target nucleic acid), and vice versa.

In various embodiments, the RNA/DNA hybrid is made in vitro using enzymatic or chemical synthetic methods such as those described herein or those described in WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999. In other embodiments, a DNA strand synthesized in vitro is complexed with an RNA strand made in vivo or in vitro before, after, or concurrent with the transformation of the DNA strand into the cell. In yet other embodiments, the dsRNA is a single circular nucleic acid containing a sense and an antisense region, or the dsRNA includes a circular nucleic acid and either a second circular nucleic acid or a linear nucleic acid (see, for example, WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999.) Exemplary circular nucleic acids include lariat structures in which the free 5′ phosphoryl group of a nucleotide becomes linked to the 2′ hydroxyl group of another nucleotide in a loop back fashion.

In other embodiments, the dsRNA includes one or more modified nucleotides in which the 2′ position in the sugar contains a halogen (such as fluorine group) or contains an alkoxy group (such as a methoxy group) which increases the half-life of the dsRNA in vitro or in vivo compared to the corresponding dsRNA in which the corresponding 2′ position contains a hydrogen or an hydroxyl group. In yet other embodiments, the dsRNA includes one or more linkages between adjacent nucleotides other than a naturally-occurring phosphodiester linkage. Examples of such linkages include phosphoramide, phosphorothioate, and phosphorodithioate linkages. The dsRNAs may also be chemically modified nucleic acid molecules as taught in U.S. Pat. No. 6,673,661. In other embodiments, the dsRNA contains one or two capped strands, as disclosed, for example, by WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999.

In other embodiments, the dsRNA can be any of the at least partially dsRNA molecules disclosed in WO 00/63364, as well as any of the dsRNA molecules described in U.S. Provisional Application 60/399,998; and U.S. Provisional Application 60/419,532, and PCT/US2003/033466, the teaching of which is hereby incorporated by reference. Any of the dsRNAs may be expressed in vitro or in vivo using the methods described herein or standard methods, such as those described in WO 00/63364.

Occupancy

In certain embodiments, antisense compounds are not expected to result in cleavage or the target nucleic acid via RNase H or to result in cleavage or sequestration through the RISC pathway. In certain such embodiments, antisense activity may result from occupancy, wherein the presence of the hybridized antisense compound disrupts the activity of the target nucleic acid. In certain such embodiments, the antisense compound may be uniformly modified or may comprise a mix of modifications and/or modified and unmodified nucleosides.

Target Nucleic Acids, Target Regions and Nucleotide Sequences

Nucleotide sequences that encode wild-type rhodopsin, without limitation, genomic sequence having the sequence set forth in GENBANK Accession No. NT_005612.16 truncated from nucleotides 35737800 to 35755500 (incorporated herein as SEQ ID NO: 1) and coding sequence having the sequence set forth in GENBANK Accession No NM_000539.3 (incorporated herein as SEQ ID NO: 3). Nucleotide sequences that encode mutant P23H rhodopsin nucleic acid have a C to A mutation at nucleotide 163 of GENBANK Accession No NM_000539.3 and is incorporated herein as SEQ ID NO: 2.

Hybridization

In some embodiments, hybridization occurs between an antisense compound disclosed herein and a P23H rhodopsin nucleic acid. The most common mechanism of hybridization involves hydrogen bonding (e.g., Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementary nucleobases of the nucleic acid molecules.

Hybridization can occur under varying conditions. Stringent conditions are sequence-dependent and are determined by the nature and composition of the nucleic acid molecules to be hybridized.

Methods of determining whether a sequence is specifically hybridizable to a target nucleic acid are well known in the art. In certain embodiments, the antisense compounds provided herein are specifically hybridizable with a P23H rhodopsin nucleic acid.

Complementarity

An antisense compound and a target nucleic acid are complementary to each other when a sufficient number of nucleobases of the antisense compound can hydrogen bond with the corresponding nucleobases of the target nucleic acid, such that a desired effect will occur (e.g., antisense inhibition of a target nucleic acid, such as a P23H rhodopsin nucleic acid).

Non-complementary nucleobases between an antisense compound and a P23H rhodopsin nucleic acid may be tolerated provided that the antisense compound remains able to specifically hybridize to a target nucleic acid. Moreover, an antisense compound may hybridize over one or more segments of a P23H rhodopsin nucleic acid such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch or hairpin structure).

In certain embodiments, the antisense compounds provided herein, or a specified portion thereof, are, or are at least, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to a P23H rhodopsin nucleic acid, a target region, target segment, or specified portion thereof. Percent complementarity of an antisense compound with a target nucleic acid can be determined using routine methods.

For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense compound which is 18 nucleobases in length having four noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J Mol. Biol., 1990, 215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649656). Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482489).

In certain embodiments, the antisense compounds provided herein, or specified portions thereof, are fully complementary (i.e. 100% complementary) to a target nucleic acid, or specified portion thereof. For example, an antisense compound may be fully complementary to a P23H rhodopsin nucleic acid, or a target region, or a target segment or target sequence thereof. As used herein, “fully complementary” means each nucleobase of an antisense compound is capable of precise base pairing with the corresponding nucleobases of a target nucleic acid. For example, a 20 nucleobase antisense compound is fully complementary to a target sequence that is 400 nucleobases long, so long as there is a corresponding 20 nucleobase portion of the target nucleic acid that is fully complementary to the antisense compound. Fully complementary can also be used in reference to a specified portion of the first and/or the second nucleic acid. For example, a 20 nucleobase portion of a 30 nucleobase antisense compound can be “fully complementary” to a target sequence that is 400 nucleobases long. The 20 nucleobase portion of the 30 nucleobase oligonucleotide is fully complementary to the target sequence if the target sequence has a corresponding 20 nucleobase portion wherein each nucleobase is complementary to the 20 nucleobase portion of the antisense compound. At the same time, the entire 30 nucleobase antisense compound may or may not be fully complementary to the target sequence, depending on whether the remaining 10 nucleobases of the antisense compound are also complementary to the target sequence.

The location of a non-complementary nucleobase may be at the 5′ end or 3′ end of the antisense compound. Alternatively, the non-complementary nucleobase or nucleobases may be at an internal position of the antisense compound. When two or more non-complementary nucleobases are present, they may be contiguous (i.e. linked) or non-contiguous. In one embodiment, a non-complementary nucleobase is located in the wing segment of a gapmer antisense oligonucleotide.

In certain embodiments, antisense compounds that are, or are up to 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length comprise no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a P23H rhodopsin nucleic acid, or specified portion thereof.

In certain embodiments, antisense compounds that are, or are up to 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length comprise no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a P23H rhodopsin nucleic acid, or specified portion thereof.

The antisense compounds provided also include those which are complementary to a portion of a target nucleic acid. As used herein, “portion” refers to a defined number of contiguous (i.e. linked) nucleobases within a region or segment of a target nucleic acid. A “portion” can also refer to a defined number of contiguous nucleobases of an antisense compound. In certain embodiments, the antisense compounds, are complementary to at least an 8 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 9 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 10 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least an 11 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 12 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 13 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 14 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 15 nucleobase portion of a target segment. Also contemplated are antisense compounds that are complementary to at least a 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleobase portion of a target segment, or a range defined by any two of these values.

Identity

The antisense compounds provided herein may also have a defined percent identity to a particular nucleotide sequence, SEQ ID NO, or compound represented by a specific Isis number, or portion thereof. As used herein, an antisense compound is identical to the sequence disclosed herein if it has the same nucleobase pairing ability. For example, a RNA which contains uracil in place of thymidine in a disclosed DNA sequence would be considered identical to the DNA sequence since both uracil and thymidine pair with adenine. Shortened and lengthened versions of the antisense compounds described herein as well as compounds having non-identical bases relative to the antisense compounds provided herein also are contemplated. The non-identical bases may be adjacent to each other or dispersed throughout the antisense compound. Percent identity of an antisense compound is calculated according to the number of bases that have identical base pairing relative to the sequence to which it is being compared.

In certain embodiments, the antisense compounds, or portions thereof, are, or are at least, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to one or more of the antisense compounds or SEQ ID NOs, or a portion thereof, disclosed herein.

In certain embodiments, a portion of the antisense compound is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid.

In certain embodiments, a portion of the antisense oligonucleotide is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid.

Modifications

A nucleoside is a base-sugar combination. The nucleobase (also known as base) portion of the nucleoside is normally a heterocyclic base moiety. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar. Oligonucleotides are formed through the covalent linkage of adjacent nucleosides to one another, to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside linkages of the oligonucleotide.

Modifications to antisense compounds encompass substitutions or changes to internucleoside linkages, sugar moieties, or nucleobases. Modified antisense compounds are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target, increased stability in the presence of nucleases, or increased inhibitory activity.

Chemically modified nucleosides may also be employed to increase the binding affinity of a shortened or truncated antisense oligonucleotide for its target nucleic acid. Consequently, comparable results can often be obtained with shorter antisense compounds that have such chemically modified nucleosides.

Modified Internucleoside Linkages

The naturally occurring internucleoside linkage of RNA and DNA is a 3′ to 5′ phosphodiester linkage. Antisense compounds having one or more modified, i.e. non-naturally occurring, internucleoside linkages are often selected over antisense compounds having naturally occurring internucleoside linkages because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.

Oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom as well as internucleoside linkages that do not have a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing linkages are well known.

In certain embodiments, antisense compounds targeted to a P23H rhodopsin nucleic acid comprise one or more modified internucleoside linkages. In certain embodiments, the modified internucleoside linkages are phosphorothioate linkages. In certain embodiments, each internucleoside linkage of an antisense compound is a phosphorothioate internucleoside linkage.

Modified Sugar Moieties

Antisense compounds can optionally contain one or more nucleosides wherein the sugar group has been modified. Such sugar modified nucleosides may impart enhanced nuclease stability, increased binding affinity, or some other beneficial biological property to the antisense compounds. In certain embodiments, nucleosides comprise chemically modified ribofuranose ring moieties. Examples of chemically modified ribofuranose rings include without limitation, addition of substitutent groups (including 5′ and 2′ substituent groups, bridging of non-geminal ring atoms to form bicyclic nucleic acids (BNA), replacement of the ribosyl ring oxygen atom with S, N(R), or C(R₁)(R₂) (R, R₁ and R₂ are each independently H, C₁-C₁₂ alkyl or a protecting group) and combinations thereof. Examples of chemically modified sugars include 2′-F-5′-methyl substituted nucleoside (see PCT International Application WO 2008/101157 Published on Aug. 21, 2008 for other disclosed 5′,2′-bis substituted nucleosides) or replacement of the ribosyl ring oxygen atom with S with further substitution at the 2′-position (see published U.S. Patent Application US2005-0130923, published on Jun. 16, 2005) or alternatively 5′-substitution of a BNA (see PCT International Application WO 2007/134181 Published on Nov. 22, 2007 wherein 4′-(CH₂)—O-2′ (LNA) is substituted with for example a 5′-methyl or a 5′-vinyl group).

Examples of nucleosides having modified sugar moieties include without limitation nucleosides comprising 5′-vinyl, 5′-methyl (R or S), 4-S, 2′-F, 2′-OCH₃, 2′-OCH₂CH₃, 2′—OCH₂CH₂F and 2′-O(CH₂)₂OCH₃ substituent groups. The substituent at the 2′ position can also be selected from allyl, amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, OCF₃, OCH₂F, O(CH₂)₂SCH₃, O(CH₂)₂—O—N(R_(m))(R_(n)), O—CH₂—C(═O)—N(R_(m))(R_(n)), and O—CH₂—C(═O)—N(R_(l))—(CH₂)₂—N(R_(m))(R_(n)), where each R_(l), R_(m) and R_(n) is, independently, H or substituted or unsubstituted C₁-C₁₀ alkyl.

As used herein, “bicyclic nucleosides” refer to modified nucleosides comprising a bicyclic sugar moiety. Examples of bicyclic nucleosides include without limitation nucleosides comprising a bridge between the 4′ and the 2′ ribosyl ring atoms. In certain embodiments, antisense compounds provided herein include one or more bicyclic nucleosides comprising a 4′ to 2′ bridge. Examples of such 4′ to 2′ bridged bicyclic nucleosides, include but are not limited to one of the formulae: 4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2′; 4′-(CH₂)₂—O-2′ (ENA); 4′-CH(CH₃)—O-2′ (also referred to as constrained ethyl or cEt) and 4′-CH(CH₂OCH₃)—O-2′ (and analogs thereof see U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008); 4′-C(CH₃)(CH₃)—O-2′ (and analogs thereof see published International Application WO/2009/006478, published Jan. 8, 2009); 4′-CH₂—N(OCH₃)-2′ (and analogs thereof see published International Application WO/2008/150729, published Dec. 11, 2008); 4′-CH₂—O—N(CH₃)-2′ (see published U.S. Patent Application US2004-0171570, published Sep. 2, 2004); 4′-CH₂—N(R)—O-2′, wherein R is H, C₁-C₁₂ alkyl, or a protecting group (see U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008); 4′-CH₂—C(H)(CH₃)-2′ (see Chattopadhyaya et al., J Org. Chem., 2009, 74, 118-134); and 4′-CH₂—C(═CH₂)-2′ (and analogs thereof see published International Application WO 2008/154401, published on Dec. 8, 2008).

Further reports related to bicyclic nucleosides can also be found in published literature (see for example: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natd. Acad. Sci. U.S.A, 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J Am. Chem. Soc., 2007, 129(26) 8362-8379; Elayadi et al., Curr. Opinion Invest. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; and Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; U.S. Pat. Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 7,034,133; 7,053,207; 7,399,845; 7,547,684; and 7,696,345; U.S. Patent Publication No. US2008-0039618; US2009-0012281; U.S. Patent Ser. Nos. 60/989,574; 61/026,995; 61/026,998; 61/056,564; 61/086,231; 61/097,787; and 61/099,844; Published PCT International applications WO 1994/014226; WO 2004/106356; WO 2005/021570; WO 2007/134181; WO 2008/150729; WO 2008/154401; and WO 2009/006478. Each of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example α-L-ribofuranose and β-D-ribofuranose (see PCT international application PCT/DK98/00393, published on Mar. 25, 1999 as WO 99/14226).

In certain embodiments, bicyclic sugar moieties of BNA nucleosides include, but are not limited to, compounds having at least one bridge between the 4′ and the 2′ position of the pentofuranosyl sugar moiety wherein such bridges independently comprises 1 or from 2 to 4 linked groups independently selected from —[C(R_(a))(R_(b))]_(n)—, C(R_(a))═C(R_(b))—, —C(R_(a))═N—, —C(═O)—, —C(═NR_(a))—, —C(═S)—, —O—, —Si(R_(a))₂—, —S(═O)_(x)—, and —N(R_(a))—;

wherein:

x is 0, 1, or 2;

n is 1, 2, 3, or 4;

each R_(a) and R_(b) is, independently, H, a protecting group, hydroxyl, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical, substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃, COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁), or sulfoxyl (S(═O)-J₁); and

each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl (C(═O)—H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl or a protecting group.

In certain embodiments, the bridge of a bicyclic sugar moiety is —[C(R_(a))(R_(b))]_(n)—, —[C(R_(a))(R_(b))]_(n)—O—, —C(R_(a)R_(b))—N(R)—O— or —C(R_(a)R_(b))—O—N(R)—. In certain embodiments, the bridge is 4′-CH₂-2′, 4′-(CH₂)₂-2′, 4′-(CH₂)₃-2′, 4′-CH₂—O-2′, 4′-(CH₂)₂—O-2′, 4′-CH₂—O—N(R)-2′ and 4′-CH₂—N(R)—O-2′- wherein each R is, independently, H, a protecting group or C₁-C₁₂ alkyl.

In certain embodiments, bicyclic nucleosides are further defined by isomeric configuration. For example, a nucleoside comprising a 4′-2′ methylene-oxy bridge, may be in the α-L configuration or in the β-D configuration. Previously, α-L-methyleneoxy (4′-CH₂—O-2′) BNA's have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

In certain embodiments, bicyclic nucleosides include, but are not limited to, (A) α-L-methyleneoxy (4′-CH₂—O-2′) BNA, (B) β-D-methylencoxy (4′-CH₂—O-2′) BNA, (C) ethylencoxy (4′-(CH₂)₂—O-2′) BNA, (D) aminooxy (4′-CH₂—O—N(R)-2′) BNA, (E) oxyamino (4′-CH₂—N(R)—O-2′) BNA, and (F) methyl(methyleneoxy) (4′-CH(CH₃)—O-2′) BNA, (G) methylene-thio (4′-CH₂—S-2′) BNA, (H) methylene-amino (4′-CH₂—N(R)-2′) BNA, (I) methyl carbocyclic (4′-CH₂—CH(CH₃)-2′) BNA, (J) propylene carbocyclic (4′-(CH₂)₃-2′) BNA and (K) vinyl BNA as depicted below:

wherein Bx is the base moiety and R is independently H, a protecting group, C₁-C₁₂ alkyl or C₁-C₂ alkoxy.

In certain embodiments, bicyclic nucleosides are provided having Formula I:

wherein:

Bx is a heterocyclic base moiety;

-Q_(a)-Q_(b)-Q_(c)- is —CH₂—N(R_(c))—CH₂—, —C(═O)—N(R_(c))—CH₂—, —CH₂—O—N(R_(c))—, —CH₂—N(R_(c))—O— or —N(R_(c))—O—CH₂;

R_(c) is C₁-C₁₂ alkyl or an amino protecting group; and

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium.

In certain embodiments, bicyclic nucleosides are provided having Formula II:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;

Z_(a) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆ alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl, acyl, substituted acyl, substituted amide, thiol or substituted thio.

In one embodiment, each of the substituted groups is, independently, mono or poly substituted with substituent groups independently selected from halogen, oxo, hydroxyl, OJ_(c), NJ_(c)J_(d), SJ_(c), N₃, OC(═X)J_(c), and NJ_(c)C(═X)NJ_(c)J_(d), wherein each J_(c), J_(d) and J_(e) is, independently, H, C₁-C₆ alkyl, or substituted C₁-C₆ alkyl and X is O or NJ_(c).

In certain embodiments, bicyclic nucleosides are provided having Formula III:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;

Z_(b) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₁-C₆ alkyl, substituted C₂-C₆ alkenyl, substituted C₂-C₆ alkynyl or substituted acyl (C(═O)—).

In certain embodiments, bicyclic nucleosides are provided having Formula IV:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;

R_(d) is C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl;

each q_(a), q_(b), q_(c) and q_(a) is, independently, H, halogen, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl, C₁-C₆ alkoxyl, substituted C₁-C₆ alkoxyl, acyl, substituted acyl, C₁-C₆ aminoalkyl or substituted C₁-C₆ aminoalkyl;

In certain embodiments, bicyclic nucleosides are provided having Formula V:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;

q_(a), q_(b), q_(e) and q_(f) are each, independently, hydrogen, halogen, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₁-C₁₂ alkoxy, substituted C₁-C₁₂ alkoxy, OJ_(j), SJ_(j), SOJ_(j), SO₂J_(j), NJ_(j)J_(k), N₃, CN, C(═O)OJ_(j), C(═O)NJ_(j)J_(k), C(═O)J_(j), O—C(═O)-NJ_(j)J_(k), N(H)C(═NH)NJ_(j)J_(k), N(H)C(═O)NJ_(j)J_(k) or N(H)C(═S)NJ_(j)J_(k);

or q_(e) and q_(f) together are ═C(q_(g))(q_(h));

q_(g) and q_(h) are each, independently, H, halogen, C₁-C₁₂ alkyl or substituted C₁-C₁₂ alkyl.

The synthesis and preparation of the methyleneoxy (4′-CH₂—O-2′) BNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil, along with their oligomerization, and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226.

Analogs of methyleneoxy (4′-CH₂—O-2′) BNA and 2′-thio-BNAs, have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). Preparation of locked nucleoside analogs comprising oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99/14226). Furthermore, synthesis of 2′-amino-BNA, a novel comformationally restricted high-affinity oligonucleotide analog has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2′-amino- and 2′-methylamino-BNA's have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has been previously reported.

In certain embodiments, bicyclic nucleosides are provided having Formula VI:

wherein:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium; each q_(i), q_(j), q_(k) and q_(l) is, independently, H, halogen, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₁-C₁₂ alkoxyl, substituted C₁-C₁₂ alkoxyl, OJ_(j), SJ_(j), SOJ_(j), SO₂J_(j), NJ_(j)J_(k), N₃, CN, C(═O)OJ_(j), C(═O)NJ_(j)J_(k), C(═O)J_(j), O—C(═O)NJ_(j)J_(k), N(H)C(═NH)NJ_(j)J_(k), N(H)C(═O)NJ_(j)J_(k) or N(H)C(═S)NJ_(j)J_(k); and

q_(i) and q_(j) or q_(i) and q_(k) together are ═C(q_(g))(q_(h)), wherein q_(g) and q_(h) are each, independently, H, halogen, C₁-C₁₂ alkyl or substituted C₁-C₁₂ alkyl.

One carbocyclic bicyclic nucleoside having a 4′-(CH₂)₃-2′ bridge and the alkenyl analog bridge 4′-CH═CH—CH₂-2′ have been described (Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J. Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (Srivastava et al., J. Am. Chem. Soc., 2007, 129(26), 8362-8379).

As used herein, “4′-2′ bicyclic nucleoside” or “4′ to 2′ bicyclic nucleoside” refers to a bicyclic nucleoside comprising a furanose ring comprising a bridge connecting two carbon atoms of the furanose ring connects the 2′ carbon atom and the 4′ carbon atom of the sugar ring.

As used herein, “monocyclic nucleosides” refer to nucleosides comprising modified sugar moieties that are not bicyclic sugar moieties. In certain embodiments, the sugar moiety, or sugar moiety analogue, of a nucleoside may be modified or substituted at any position.

As used herein, “2′-modified sugar” means a furanosyl sugar modified at the 2′ position. In certain embodiments, such modifications include substituents selected from: a halide, including, but not limited to substituted and unsubstituted alkoxy, substituted and unsubstituted thioalkyl, substituted and unsubstituted amino alkyl, substituted and unsubstituted alkyl, substituted and unsubstituted allyl, and substituted and unsubstituted alkynyl. In certain embodiments, 2′ modifications are selected from substituents including, but not limited to: O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)F, O(CH₂)_(n)ONH₂, OCH₂C(═O)N(H)CH₃, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from 1 to about 10. Other 2′-substituent groups can also be selected from: C₁-C₁₂ alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, F, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving pharmacokinetic properties, or a group for improving the pharmacodynamic properties of an antisense compound, and other substituents having similar properties. In certain embodiments, modified nucleosides comprise a 2′-MOE side chain (Baker et al., J. Biol. Chem., 1997, 272, 11944-12000). Such 2′-MOE substitution have been described as having improved binding affinity compared to unmodified nucleosides and to other modified nucleosides, such as 2′-O-methyl, O-propyl, and O-aminopropyl. Oligonucleotides having the 2′-MOE substituent also have been shown to be antisense inhibitors of gene expression with promising features for in vivo use (Martin, Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides, 1997, 16, 917-926).

As used herein, a “modified tetrahydropyran nucleoside” or “modified THP nucleoside” means a nucleoside having a six-membered tetrahydropyran “sugar” substituted in for the pentofuranosyl residue in normal nucleosides (a sugar surrogate). Modified THP nucleosides include, but are not limited to, what is referred to in the art as hexitol nucleic acid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (INA) (see Leumann, Bioorg. Med. Chem., 2002, 10, 841-854) or fluoro HNA (F-HNA) having a tetrahydropyran ring system as illustrated below:

In certain embodiments, sugar surrogates are selected having Formula VII:

wherein independently for each of said at least one tetrahydropyran nucleoside analog of Formula VII:

Bx is a heterocyclic base moiety;

T_(a) and T_(b) are each, independently, an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound or one of T_(a) and T_(b) is an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound and the other of T_(a) and T_(b) is H, a hydroxyl protecting group, a linked conjugate group or a 5′ or 3′-terminal group;

q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl or substituted C₂-C₆ alkynyl; and each of R₁ and R₂ is selected from hydrogen, hydroxyl, halogen, substituted or unsubstituted alkoxy, NJ₁J₂, SJ₁, N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein X is O, S or NJ₁ and each J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl.

In certain embodiments, the modified THP nucleosides of Formula VII are provided wherein q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. In certain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is other than H. In certain embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is methyl. In certain embodiments, THP nucleosides of Formula VII are provided wherein one of R₁ and R₂ is fluoro. In certain embodiments, R₁ is fluoro and R₂ is H; R₁ is methoxy and R₂ is H, and R₁ is methoxyethoxy and R₂ is H.

In certain embodiments, sugar surrogates comprise rings having more than 5 atoms and more than one heteroatom. For example nucleosides comprising morpholino sugar moieties and their use in oligomeric compounds has been reported (see for example: Braasch et al., Biochemistry, 2002, 41, 4503-4510; and U.S. Pat. Nos. 5,698,685; 5,166,315; 5,185,444; and 5,034,506). As used here, the term “morpholino” means a sugar surrogate having the following formula:

In certain embodiments, morpholinos may be modified, for example by adding or altering various substituent groups from the above morpholino structure. Such sugar surrogates are referred to herein as “modified morpholinos.”

Combinations of modifications are also provided without limitation, such as 2′-F-5′-methyl substituted nucleosides (see PCT International Application WO 2008/101157 published on Aug. 21, 2008 for other disclosed 5′, 2′-bis substituted nucleosides) and replacement of the ribosyl ring oxygen atom with S and further substitution at the 2′-position (see published U.S. Patent Application US2005-0130923, published on Jun. 16, 2005) or alternatively 5′-substitution of a bicyclic nucleic acid (see PCT International Application WO 2007/134181, published on Nov. 22, 2007 wherein a 4′-CH₂—O-2′ bicyclic nucleoside is further substituted at the 5′ position with a 5′-methyl or a 5′-vinyl group). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (see, e.g., Srivastava et al., J Am. Chem. Soc. 2007, 129(26), 8362-8379).

In certain embodiments, antisense compounds comprise one or more modified cyclohexenyl nucleosides, which is a nucleoside having a six-membered cyclohexenyl in place of the pentofuranosyl residue in naturally occurring nucleosides. Modified cyclohexenyl nucleosides include, but are not limited to those described in the art (see for example commonly owned, published PCT Application WO 2010/036696, published on Apr. 10, 2010, Robeyns et al., J. Am. Chem. Soc., 2008, 130(6), 1979-1984; Horvith et al., Tetrahedron Letters, 2007, 48, 3621-3623; Nauwelaerts et al., J. Am. Chem. Soc., 2007, 129(30), 9340-9348; Gu et al., Nucleosides, Nucleotides & Nucleic Acids, 2005, 24(5-7), 993-998; Nauwelaerts et al., Nucleic Acids Research, 2005, 33(8), 2452-2463; Robeyns et al., Acta Crystallographica, Section F: Structural Biology and Crystallization Communications, 2005, F61(6), 585-586; Gu et al., Tetrahedron, 2004, 60(9), 2111-2123; Gu et al., Oligonucleotides, 2003, 13(6), 479-489; Wang et al., J. Org. Chem., 2003, 68, 4499-4505; Verbeure et al., Nucleic Acids Research, 2001, 29(24), 4941-4947; Wang et al., J. Org. Chem., 2001, 66, 8478-82; Wang et al., Nucleosides, Nucleotides & Nucleic Acids, 2001, 20(4-7), 785-788; Wang et al., J. Am. Chem., 2000, 122, 8595-8602; Published PCT application, WO 06/047842; and Published PCT Application WO 01/049687; the text of each is incorporated by reference herein, in their entirety). Certain modified cyclohexenyl nucleosides have Formula X.

wherein independently for each of said at least one cyclohexenyl nucleoside analog of Formula X:

Bx is a heterocyclic base moiety;

T₃ and T₄ are each, independently, an internucleoside linking group linking the cyclohexenyl nucleoside analog to an antisense compound or one of T₃ and T₄ is an internucleoside linking group linking the tetrahydropyran nucleoside analog to an antisense compound and the other of T₃ and T₄ is H, a hydroxyl protecting group, a linked conjugate group, or a 5′- or 3′-terminal group; and

q₁, q₂, q₃, q₄, q₅, q₆, q₇, q₈ and q₉ are each, independently, H, C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl or other sugar substituent group.

As used herein, “2′-modified” or “2′-substituted” refers to a nucleoside comprising a sugar comprising a substituent at the 2′ position other than H or OH. 2′-modified nucleosides, include, but are not limited to, bicyclic nucleosides wherein the bridge connecting two carbon atoms of the sugar ring connects the 2′ carbon and another carbon of the sugar ring; and nucleosides with non-bridging 2′substituents, such as allyl, amino, azido, thio, O-allyl, O—C₁-C₁₀ alkyl, —OCF₃, O—(CH₂)₂O—CH₃, 2′—O(CH₂)₂SCH₃, O—(CH₂)₂—O—N(R_(m))(R_(n)), or O—CH₂—C(═O)—N(R_(m))(R_(n)), where each R_(m) and R_(n) is, independently, H or substituted or unsubstituted C₁-C₁₀ alkyl. 2′-modified nucleosides may further comprise other modifications, for example at other positions of the sugar and/or at the nucleobase.

As used herein, “2′-F” refers to a nucleoside comprising a sugar comprising a fluoro group at the 2′ position of the sugar ring.

As used herein, “2′-OMe” or “2′-OCH₃” or “2′-O-methyl” each refers to a nucleoside comprising a sugar comprising an —OCH₃ group at the 2′ position of the sugar ring.

As used herein, “oligonucleotide” refers to a compound comprising a plurality of linked nucleosides. In certain embodiments, one or more of the plurality of nucleosides is modified. In certain embodiments, an oligonucleotide comprises one or more ribonucleosides (RNA) and/or deoxyribonucleosides (DNA).

Many other bicyclo and tricyclo sugar surrogate ring systems are also known in the art that can be used to modify nucleosides for incorporation into antisense compounds (see for example review article: Leumann, Bioorg. Med. Chem., 2002, 10, 841-854). Such ring systems can undergo various additional substitutions to enhance activity.

Methods for the preparations of modified sugars are well known to those skilled in the art. Some representative U.S. patents that teach the preparation of such modified sugars include without limitation, U.S.: 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,670,633; 5,700,920; 5,792,847 and 6,600,032 and International Application PCT/US2005/019219, filed Jun. 2, 2005 and published as WO 2005/121371 on Dec. 22, 2005, and each of which is herein incorporated by reference in its entirety.

In nucleotides having modified sugar moieties, the nucleobase moieties (natural, modified or a combination thereof) are maintained for hybridization with an appropriate nucleic acid target.

In certain embodiments, antisense compounds comprise one or more nucleosides having modified sugar moieties. In certain embodiments, the modified sugar moiety is 2′-MOE. In certain embodiments, the 2′-MOE modified nucleosides are arranged in a gapmer motif. In certain embodiments, the modified sugar moiety is a bicyclic nucleoside having a (4′-CH(CH₃)—O-2′) bridging group. In certain embodiments, the (4′-CH(CH₃)—O-2′) modified nucleosides are arranged throughout the wings of a gapmer motif.

Modified Nucleobases

Nucleobase (or base) modifications or substitutions are structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic unmodified nucleobases. Both natural and modified nucleobases are capable of participating in hydrogen bonding. Such nucleobase modifications can impart nuclease stability, binding affinity or some other beneficial biological property to antisense compounds. Modified nucleobases include synthetic and natural nucleobases such as, for example, 5-methylcytosine (5-me-C). Certain nucleobase substitutions, including 5-methylcytosine substitutions, are particularly useful for increasing the binding affinity of an antisense compound for a target nucleic acid. For example, 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278).

Additional modified nucleobases include 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C═C—CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

Heterocyclic base moieties can also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Nucleobases that are particularly useful for increasing the binding affinity of antisense compounds include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2 aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.

In certain embodiments, antisense compounds targeted to a P23H rhodopsin nucleic acid comprise one or more modified nucleobases. In certain embodiments, shortened or gap-widened antisense oligonucleotides targeted to a P23H rhodopsin nucleic acid comprise one or more modified nucleobases. In certain embodiments, the modified nucleobase is 5-methylcytosine. In certain embodiments, each cytosine is a 5-methylcytosine.

Conjugated Antisense Compounds

Antisense compounds may be covalently linked to one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the resulting antisense oligonucleotides. Typical conjugate groups include cholesterol moieties and lipid moieties. Additional conjugate groups include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.

Antisense compounds can also be modified to have one or more stabilizing groups that are generally attached to one or both termini of antisense compounds to enhance properties such as, for example, nuclease stability. Included in stabilizing groups are cap structures. These terminal modifications protect the antisense compound having terminal nucleic acid from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5′-terminus (5′-cap), or at the 3′-terminus (3′-cap), or can be present on both termini. Cap structures are well known in the art and include, for example, inverted deoxy abasic caps. Further 3′ and 5′-stabilizing groups that can be used to cap one or both ends of an antisense compound to impart nuclease stability include those disclosed in WO 03/004602 published on Jan. 16, 2003.

In certain embodiments, antisense compounds, including, but not limited to those particularly suited for use as ssRNA, are modified by attachment of one or more conjugate groups. In general, conjugate groups modify one or more properties of the attached oligonucleotide, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, cellular distribution, cellular uptake, charge and clearance. Conjugate groups are routinely used in the chemical arts and are linked directly or via an optional conjugate linking moiety or conjugate linking group to a parent compound such as an oligonucleotide. Conjugate groups includes without limitation, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins and dyes. Certain conjugate groups have been described previously, for example: cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), athioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., do-decan-diol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).

For additional conjugates including those useful for ssRNA and their placement within antisense compounds, see e.g., U.S. Application No. 61/583,963.

In Vitro Testing of Antisense Oligonucleotides

Described herein are methods for treatment of cells with antisense oligonucleotides, which can be modified appropriately for treatment with other antisense compounds.

Cells may be treated with antisense oligonucleotides when the cells reach approximately 60-80% confluency in culture.

One reagent commonly used to introduce antisense oligonucleotides into cultured cells includes the cationic lipid transfection reagent LIPOFECTIN (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotides may be mixed with LIPOFECTIN in OPTI-MEM 1 (Invitrogen, Carlsbad, Calif.) to achieve the desired final concentration of antisense oligonucleotide and a LIPOFECTIN concentration that may range from 2 to 12 ug/mL per 100 nM antisense oligonucleotide.

Another reagent used to introduce antisense oligonucleotides into cultured cells includes LIPOFECTAMINE (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotide is mixed with LIPOFECTAMINE in OPTI-MEM 1 reduced serum medium (Invitrogen, Carlsbad, Calif.) to achieve the desired concentration of antisense oligonucleotide and a LIPOFECTAMINE concentration that may range from 2 to 12 ug/mL per 100 nM antisense oligonucleotide.

Another technique used to introduce antisense oligonucleotides into cultured cells includes electroporation.

Yet another technique used to introduce antisense oligonucleotides into cultured cells includes free uptake of the oligonucleotides by the cells.

Cells are treated with antisense oligonucleotides by routine methods. Cells may be harvested 16-24 hours after antisense oligonucleotide treatment, at which time RNA or protein levels of target nucleic acids are measured by methods known in the art and described herein. In general, when treatments are performed in multiple replicates, the data are presented as the average of the replicate treatments.

The concentration of antisense oligonucleotide used varies from cell line to cell line. Methods to determine the optimal antisense oligonucleotide concentration for a particular cell line are well known in the art. Antisense oligonucleotides are typically used at concentrations ranging from 1 nM to 300 nM when transfected with LIPOFECTAMINE. Antisense oligonucleotides are used at higher concentrations ranging from 625 to 20,000 nM when transfected using electroporation.

RNA Isolation

RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well known in the art. RNA is prepared using methods well known in the art, for example, using the TRIZOL Reagent (Invitrogen, Carlsbad, Calif.) according to the manufacturer's recommended protocols.

Compositions and Methods for Formulating Pharmaceutical Compositions

Antisense compounds may be admixed with pharmaceutically acceptable active or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions are dependent upon a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.

An antisense compound targeted to P23H rhodopsin nucleic acid can be utilized in pharmaceutical compositions by combining the antisense compound with a suitable pharmaceutically acceptable diluent or carrier. In certain embodiments, a pharmaceutically acceptable diluent is water, such as sterile water suitable for injection. Accordingly, in one embodiment, employed in the methods described herein is a pharmaceutical composition comprising an antisense compound targeted to P23H rhodopsin nucleic acid and a pharmaceutically acceptable diluent. In certain embodiments, the pharmaceutically acceptable diluent is water. In certain embodiments, the antisense compound is an antisense oligonucleotide provided herein.

Pharmaceutical compositions comprising antisense compounds encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other oligonucleotide which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

A prodrug can include the incorporation of additional nucleosides at one or both ends of an antisense compound which are cleaved by endogenous nucleases within the body, to form the active antisense compound.

In certain embodiments, the compounds or compositions further comprise a pharmaceutically acceptable carrier or diluent.

EXAMPLES

The Examples below describe the screening process to identify lead compounds targeted to P23H mutant rhodopsin. Out of over 400 antisense oligonucleotides that were screened, ISIS 564426, ISIS 664844, ISIS 664867, and ISIS 664884 emerged as the top lead compounds. In particular, ISIS 664844 exhibited the best combination of properties in terms of potency, tolerability, and selectivity for P23H rhodopsin out of over 400 antisense oligonucleotides.

Non-Limiting Disclosure and Incorporation by Reference

While certain compounds, compositions and methods described herein have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the references recited in the present application is incorporated herein by reference in its entirety.

Although the sequence listing accompanying this filing identifies each sequence as either “RNA” or “DNA” as required, in reality, those sequences may be modified with any combination of chemical modifications. One of skill in the art will readily appreciate that such designation as “RNA” or “DNA” to describe modified oligonucleotides is, in certain instances, arbitrary. For example, an oligonucleotide comprising a nucleoside comprising a 2′-OH sugar moiety and a thymine base could be described as a DNA having a modified sugar (2′-OH for the natural 2′-H of DNA) or as an RNA having a modified base (thymine (methylated uracil) for natural uracil of RNA).

Accordingly, nucleic acid sequences provided herein, including, but not limited to those in the sequence listing, are intended to encompass nucleic acids containing any combination of natural or modified RNA and/or DNA, including, but not limited to such nucleic acids having modified nucleobases. By way of further example and without limitation, an oligonucleotide having the nucleobase sequence “ATCGATCG” encompasses any oligonucleotides having such nucleobase sequence, whether modified or unmodified, including, but not limited to, such compounds comprising RNA bases, such as those having sequence “AUCGAUCG” and those having some DNA bases and some RNA bases such as “AUCGATCG”.

Example 1: Design and In Vitro Screening of Human Rhodopsin

Antisense oligonucleotides were designed targeting human wild-type or P23H mutant rhodopsin nucleic acid and were tested for their effects on rhodopsin mRNA in vitro. Cell lines either expressing the entire rhodopsin genomic sequence or transfected with a mini gene were used in the assays. The cell lines are described further in the experiments in the Examples below. Two hundred and twelve MOE gapmers, with various motifs (5-10-5, 6-8-6, 7-6-7, 4-10-4, 5-8-5, 6-6-6, 3-10-3, 4-8-4, and 5-6-5) were tested in vitro for potency. Two hundred and two cEt gapmers, as well as gapmers with cEt and MOE modifications, were tested in vitro for potency. Of all these tested gapmers, 104 gapmers were tested in in vitro dose response assays.

The newly designed chimeric antisense oligonucleotides in the Table below were designed as 3-10-3 cEt gapmers. The gapmers are 16 nucleosides in length, wherein the central gap segment comprises ten 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising three nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a cEt modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines. “Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. ‘Mismatch’ indicates the number of mismatches the oligonucleotide sequence may have with the genomic sequence. Mismatches of more than 1 were not considered. The gapmers are targeted to the human rhodopsin genomic sequence, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NT_005612.16 truncated from nucleotides 35737800 to 35755500 or to the P23H rhodopsin mutant sequence having a cytosine to adenine substitution at position 163 of GENBANK Accession No. NM_000539.3; designated herein as SEQ ID NO:2 representing the mutant sequence), or both sequences. ‘n/a’ indicates that the particular oligonucleotide had more than one mismatch with the target gene sequence. The gapmers are presented in the Table below.

TABLE 1 3-10-3 gapmers targeting wild-type Rho (SEQ ID NO: 1) and P23H Rho (SEQ ID NO: 2) SEQ ID SEQ ID SEQ ID SEQ ID NO: 1 NO: 1 Mismatch NO: 2 NO: 2 SEQ ISIS Start Stop with SEQ ID Start Stop ID NO Site Site NO: 1 Sequence Site Site NO 564387 4979 4994 1 AAGTGGCTGCGTACCA 151 166 11 564389 4983 4998 1 CTCGAAGTGGCTGCGT 155 170 12 564424 4977 4992 1 GTGGCTGCGTACCACA 149 164 13 564425 4981 4996 1 CGAAGTGGCTGCGTAC 153 168 14 564426 4985 5000 1 TACTCGAAGTGGCTGC 157 172 15 564283 4898 4913 0 CTTGTGGCTGACCCGT 70 85 65 564284 4935 4950 0 GAAGTTAGGGCCTTCT 107 122 66 564393 6112 6127 0 CAGCAGAGATATTCCT n/a n/a 67 564430 8414 8429 0 CAGGTAGGGAGACCCT n/a n/a 68 564433 8963 8978 0 CACCCGCAGTAGGCAC n/a n/a 69 564431 9444 9459 0 AGGAAATTGACTTGCC n/a n/a 70 564338 9851 9866 0 AGCAGAGGCCTCATCG 1085 1100 71 564342 9909 9924 0 GAGTCCTAGGCAGGTC 1143 1158 72 564299 10092 10107 0 GGTGGATGTCCCTTCT 1326 1341 73 564356 10192 10207 0 AAAGCAAGAATCCTCG 1426 1441 74 564307 10517 10532 0 GCTATTTACAAAGTGC 1751 1766 75 564370 10539 10554 0 ACTAGAATCTGTACAG 1773 1788 76 564372 10578 10593 0 ATTAACTAGTTACATT 1812 1827 77 564315 10654 10669 0 CCAAGGTTGGGTGAAA 1888 1903 78 564388 10757 10772 0 GGTCTGATGACTGCAT 1991 2006 79 564325 10791 10806 0 TTCACCGTCCCCCTCC 2025 2040 80 564329 10824 10839 0 AGGCCCAATCTCACCC 2058 2073 81 564399 10930 10945 0 AAGAGCAGGTGGCTTC 2164 2179 82 564349 11048 11063 0 CTAAGCTCTTCGAGAT 2282 2297 83 564363 11237 11252 0 AGCAGTTACTGAGGCA 2471 2486 84 564373 11359 11374 0 CAAAACCCACCACCGT 2593 2608 85 564381 11456 11471 0 TTGGCTCTGCTCATTG 2690 2705 86 564422 11465 11480 0 CTGTGCTGCTTGGCTC 2699 2714 87

Gapmers were tested at various doses in HEK-293 cells. HEK-293 cells expressing the the human genomic P23H rhodopsin sequence as a stable transfectant were used for these assays. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cells were plated at a density of 20,000 cells per well and transfected using electroporation with antisense oligonucleotide, as specified in the Tables below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and rhodopsin mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3374 (forward sequence GGAGGTCAACAACGAGTCTTTTG, designated herein as SEQ ID NO: 5; reverse sequence GGCCTCCTTGACGGTGAA, designated herein as SEQ ID NO: 6; probe sequence TTATCATCTTTTTCTGCTATGGGCAGCTCG, designated herein as SEQ ID NO: 7) was used to measure mRNA levels. Rhodopsin mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of rhodopsin, relative to untreated control cells.

TABLE 2 Dose Response Inhibition of P23H RHO mRNA levels by 3-10-3 cEt gapmers targeted to SEQ ID NO: 2 1.25 2.50 5.00 10.00 20.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 564283 12 25 35 22 40 >20 564284 0 0 3 0 44 >20 564299 29 30 64 31 11 >20 564307 26 0 28 21 17 >20 564315 10 16 28 16 21 >20 564325 44 52 66 81 86 2 564329 0 2 10 16 0 >20 564349 0 0 0 0 1 >20 564363 17 0 20 13 31 >20 564373 19 17 10 29 38 >20 564381 16 18 34 33 42 >20 564387 19 26 39 42 76 7 564389 35 37 39 18 50 >20 564393 17 7 20 38 40 >20

TABLE 3 Dose Response Inhibition of P23H RHO mRNA levels by 3-10-3 cEt gapmers targeted to SEQ ID NO: 2 1.25 2.50 5.00 10.00 20.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 564338 0 20 35 19 25 >20 564342 32 31 40 0 36 >20 564356 21 18 31 13 0 >20 564370 0 0 15 10 17 >20 564372 0 0 0 0 23 >20 564388 0 0 20 27 2 >20 564399 9 0 24 30 35 >20 564422 4 0 20 17 51 9 564424 5 0 21 0 0 >20 564425 0 14 17 14 31 >20 564426 1 14 17 21 33 >20 564430 0 0 17 25 5 >20 564431 26 29 43 52 43 >20 564433 0 0 13 4 0 >20

Example 2: Design of Antisense Oligonucleotides with Deoxy, 2′-Alpha-Fluoro, and cEt Chemistry

Additional antisense oligonucleotides were designed with the same sequence as ISIS 564387 but with different chemistry. The new antisense oligonucleotides were designed as deoxy, 2′-alpha-fluoro and cEt oligonucleotides.

The ‘Chemistry’ column of the Table below presents chemical modifications in the oligonucleotide, including the position of the sugar modifications, wherein ‘e’ indicates a MOE modification, ‘k’ indicates a cEt modification, d indicates a deoxyribose sugar, and f indicates a 2′-alpha-fluoro modification; ‘mC’ indicates 5-methycytosine; ‘A’, ‘C’, ‘T’, ‘G’, and ‘U’ represent the standard nucleotide notations. All the oligonucleotides are 15 or 16 nucleosides in length. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. “Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. The antisense oligonucleotides were designed to target the mutant sequence (SEQ ID NO:2). The oligonucleotides are presented in the Table below. All the oligonucleotides target nucleotides 151-166 of SEQ ID NO: 2.

TABLE 4 Antisense oligonucleotides targeting the mutant P23H rhodopsin gene (SEQ ID NO: 2) IsisNo Chemistry SEQ ID NO 564387 Aks Aks Gks Tds Gds Gds mCds Tds Gds 11 mCds Gds Tds Ads mCks mCks Ak 598202 Aks Aks Gks Ufs Gds Gds mCds Tds Gds 11 mCds Gds Tds Ads mCks mCks Ak 598203 Aks Aks Gks Tds Gfs Gds mCds Tds Gds 11 mCds Gds Tds Ads mCks mCks Ak 598204 Aks Aks Gks Tds Gds Gfs mCds Tds Gds 11 mCds Gds Tds Ads mCks mCks Ak 598205 Aks Aks Gks Tds Gds Gds Cfs Tds Gds 11 mCds Gds Tds Ads mCks mCks Ak 598206 Aks Aks Gks Tds Gds Gds mCds Ufs Gds 11 mCds Gds Tds Ads mCks mCks Ak 598207 Aks Aks Gks Tds Gds Gds mCds Tds Gfs 11 mCds Gds Tds Ads mCks mCks Ak 598208 Aks Aks Gks Tds Gds Gds mCds Tds Gds 11 Cfs Gds Tds Ads mCks mCks Ak 598209 Aks Aks Gks Tds Gds Gds mCds Tds Gds 11 mCds Gfs Tds Ads mCks mCks Ak 598210 Aks Aks Gks Tds Gds Gds mCds Tds Gds 11 mCds Gds Ufs Ads mCks mCks Ak 598211 Aks Aks Gks Tds Gds Gds mCds Tds Gds 11 mCds Gds Tds Afs mCks mCks Ak

Example 3: Antisense Inhibition of Mutant P23H Human Rhodopsin

Additional antisense oligonucleotides were designed targeting the sequence region around the P23H mutation site of the rhodopsin gene and were tested for their effects on mutant rhodopsin mRNA in vitro. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cultured HEK293 transfected with a SOD1 minigene containing mutant P23H rhodopsin were used in this assay.

The SOD1 minigene contains the unspliced sequence of SOD1 exon 4, intron 4, and exon 5, with a human rhodopsin sequence with the mutation at P23H. Each sequence was cloned into pcDNA4/TO at HindIII/EcoRI site.

HEK-293 cells with the SOD1 minigene containing mutant P23H rhodopsin were transfected using electroporation with 5 μM or 20 μM antisense oligonucleotide. ISIS 564425, described in the study above, was also included in the assay. After a treatment period of approximately 24 hours, RNA was isolated from the cells and rhodopsin mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS4220 (forward sequence CACTATAGGGAGACCCAAGC, designated herein as SEQ ID NO: 8; reverse sequence CTGCTTTTTCATGGACCACCA, designated herein as SEQ ID NO: 9; probe sequence CAAAGATGGTGTGGCCG, designated herein as SEQ ID NO: 10), which is targeted to the P23H site, was used to measure mRNA levels. Rhodopsin mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of rhodopsin, relative to untreated control cells.

The newly designed chimeric antisense oligonucleotides in the Table below were designed as 3-10-3 cEt gapmers, 3-9-3 cEt gapmers, deoxy, MOE and cEt oligonucleotides, or deoxy, 2′-alpha-fluoro and cEt oligonucleotides. The ‘Chemistry’ column of the Table below presents chemical modifications in the oligonucleotide, including the position of the sugar modifications, wherein ‘e’ indicates a MOE modification, ‘k’ indicates a cEt modification, d indicates a deoxyribose sugar, and ‘f’ indicates a 2′-alpha-fluoro modification; ‘mC’ indicates 5-methycytosine; ‘A’, ‘C’, ‘T’, ‘G’, and ‘U’ represent the standard nucleotide notations. All the oligonucleotides are 15 or 16 nucleosides in length. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. “Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. The antisense oligonucleotides were designed to target the mutant P23H sequence (SEQ ID NO:2). The oligonucleotides are presented in the Table below.

TABLE 5 Inhibition of P23H rhodopsin mRNA by antisense oligonucleotides targeting the mutant rhodopsin gene (SEQ ID NO: 2) SEQ ID SEQ ID NO: 2 NO: 2 % % SEQ ISIS Start Stop inhibition inhibition ID NO Site Site Chemistry Sequence (5 μM) (20 μM) NO 564425 153 168 mCkGkAkAdGdTdGdGdmCdT CGAAGTGGCTGCGTAC 62 72 14 dGdmCdGdTkAkmCk 598206 151 166 AkAkGkTdGdGdmCdUfGdmCd AAGTGGCUGCGTACCA 55 89 62 GdTdAdmCkmCkAk 664823 149 164 GkTdGdGdmCdTdGdmCdGdTd GTGGCTGCGTACCACA  6 52 13 AdmCkmCeAkmCeAk 664824 150 165 AkGdTdGdGdmCdTdGdmCdG AGTGGCTGCGTACCAC 50 77 16 dTdAkmCemCkAemCk 664825 151 166 AkAdGdTdGdGdmCdTdGdmC AAGTGGCTGCGTACCA 39 62 11 dGdTkAcmCkmCcAk 664826 152 167 GkAdAdGdTdGdGdmCdTdGd GAAGTGGCTGCGTACC 46 66 17 mCdGkTeAkmCemCk 664827 153 168 mCkGdAdAdGdTdGdGdmCdT CGAAGTGGCTGCGTAC 53 52 14 dGdmCkGeTkAemCk 664828 154 169 TkmCdGdAdAdGdTdGdGdmC TCGAAGTGGCTGCGTA 40 66 18 dTdGkmCeGkTeAk 664829 155 170 mCkTdmCdGdAdAdGdTdGdG CTCGAAGTGGCTGCGT 35 59 12 dmCdTkGemCkGeTk 664830 156 171 AkmCdTdmCdGdAdAdGdTdG ACTCGAAGTGGCTGCG 38 67 19 dGdmCkTeGkmCeGk 664831 157 172 TkAdmCdTdmCdGdAdAdGdTd TACTCGAAGTGGCTGC 39 63 15 GdGkmCeTkGemCk 664832 158 173 GkTdAdmCdTdmCdGdAdAdG GTACTCGAAGTGGCTG 10 51 20 dTdGkGemCkTeGk 664833 159 174 GkGdTdAdmCdTdmCdGdAdA GGTACTCGAAGTGGCT 57 68 21 dGdTkGcGkmCcTk 664834 149 164 GkTkGdGdmCdTdGdmCdGdTd GTGGCTGCGTACCACA 33 50 13 AdmCdmCkAemCkAe 664835 150 165 AkGkTdGdGdmCdTdGdmCdG AGTGGCTGCGTACCAC 39 75 16 dTdAdmCkmCeAkmCe 664836 151 166 AkAkGdTdGdGdmCdTdGdmC AAGTGGCTGCGTACCA 56 76 11 dGdTdAkmCemCkAe 664837 152 167 GkAkAdGdTdGdGdmCdTdGd GAAGTGGCTGCGTACC 48 72 17 mCdGdTkAcmCkmCc 664838 153 168 mCkGkAdAdGdTdGdGdmCdT CGAAGTGGCTGCGTAC 38 84 14 dGdmCdGkTeAkmCe 664839 154 169 TkmCkGdAdAdGdTdGdGdmC TCGAAGTGGCTGCGTA 49 72 18 dTdGdmCkGeTkAe 664840 155 170 mCkTkmCdGdAdAdGdTdGdG CTCGAAGTGGCTGCGT 55 61 12 dmCdTdGkmCeGkTe 664841 156 171 AkmCkTdmCdGdAdAdGdTdG ACTCGAAGTGGCTGCG 47 68 19 dGdmCdTkGcmCkGc 664842 157 172 TkAkmCdTdmCdGdAdAdGdTd TACTCGAAGTGGCTGC 48 72 15 GdGdmCkTeGkmCe 664843 158 173 GkTkAdmCdTdmCdGdAdAdG GTACTCGAAGTGGCTG 64 73 20 dTdGdGkmCeTkGe 664844 159 174 GkGkTdAdmCdTdmCdGdAdA GGTACTCGAAGTGGCT 61 64 21 dGdTdGkGemCkTe 664845 149 164 GkTkGdGdmCdTdGdmCdGdTd GTGGCTGCGTACCACA 10 45 13 AdmCkmCeAkmCeAk 664846 150 165 AkGkTdGdGdmCdTdGdmCdG AGTGGCTGCGTACCAC 58 69 16 dTdAkmCemCkAemCk 664847 151 166 AkAkGdTdGdGdmCdTdGdmC AAGTGGCTGCGTACCA 41 56 11 dGdTkAemCkmCeAk 664848 152 167 GkAkAdGdTdGdGdmCdTdGd GAAGTGGCTGCGTACC 49 66 17 mCdGkTeAkmCemCk 664849 153 168 mCkGkAdAdGdTdGdGdmCdT CGAAGTGGCTGCGTAC 57 72 14 dGdmCkGeTkAemCk 664850 154 169 TkmCkGdAdAdGdTdGdGdmC TCGAAGTGGCTGCGTA 42 65 18 dTdGkmCeGkTeAk 664851 155 170 mCkTkmCdGdAdAdGdTdGdG CTCGAAGTGGCTGCGT 20 59 12 dmCdTkGemCkGeTk 664852 156 171 AkmCkTdmCdGdAdAdGdTdG ACTCGAAGTGGCTGCG 42 57 19 dGdmCkTeGkmCeGk 664853 157 172 TkAkmCdTdmCdGdAdAdGdTd TACTCGAAGTGGCTGC 43 67 15 GdGkmCeTkGemCk 664854 158 173 GkTkAdmCdTdmCdGdAdAdG GTACTCGAAGTGGCTG 34 55 20 dTdGkGemCkTeGk 664855 159 174 GkGkTdAdmCdTdmCdGdAdA GGTACTCGAAGTGGCT 48 63 21 dGdTkGeGkmCeTk 664856 149 164 GkTkGkGdmCdTdGdmCdGdTd GTGGCTGCGTACCACA 20 43 13 AdmCdmCkAcmCkAc 664857 150 165 AkGkTkGdGdmCdTdGdmCdG AGTGGCTGCGTACCAC  0 37 16 dTdAdmCkmCeAkmCe 664858 151 166 AkAkGkTdGdGdmCdTdGdmC AAGTGGCTGCGTACCA 52 81 11 dGdTdAkmCemCkAe 664859 152 167 GkAkAkGdTdGdGdmCdTdGd GAAGTGGCTGCGTACC 52 74 17 mCdGdTkAemCkmCe 664860 153 168 mCkGkAkAdGdTdGdGdmCdT CGAAGTGGCTGCGTAC 56 74 14 dGdmCdGkTeAkmCe 664861 154 169 TkmCkGkAdAdGdTdGdGdmC TCGAAGTGGCTGCGTA 33 58 18 dTdGdmCkGeTkAe 664862 155 170 mCkTkmCkGdAdAdGdTdGdG CTCGAAGTGGCTGCGT 39 64 12 dmCdTdGkmCeGkTe 664863 156 171 AkmCkTkmCdGdAdAdGdTdG ACTCGAAGTGGCTGCG 45 70 19 dGdmCdTkGemCkGe 664864 157 172 TkAkmCkTdmCdGdAdAdGdTd TACTCGAAGTGGCTGC 49 69 15 GdGdmCkTeGkmCe 664865 158 173 GkTkAkmCdTdmCdGdAdAdG GTACTCGAAGTGGCTG 54 67 20 dTdGdGkmCeTkGe 664866 159 174 GkGkTkAdmCdTdmCdGdAdA GGTACTCGAAGTGGCT 54 64 21 dGdTdGkGemCkTe 664867 157 172 TkAkmCkUfmCdGdAdAdGdTd TACUCGAAGTGGCTGC 66 76 64 GdGdmCdTkGkmCk 664868 157 172 TkAkmCkTdCfGdAdAdGdTdG TACTCGAAGTGGCTGC 54 69 15 dGdmCdTkGkmCk 664869 157 172 TkAkmCkTdmCdGfAdAdGdTd TACTCGAAGTGGCTGC 53 69 15 GdGdmCdTkGkmCk 664870 157 172 TkAkmCkTdmCdGdAfAdGdTd TACTCGAAGTGGCTGC 54 69 15 GdGdmCdTkGkmCk 664871 157 172 TkAkmCkTdmCdGdAdAfGdTd TACTCGAAGTGGCTGC 45 68 15 GdGdmCdTkGkmCk 664872 157 172 TkAkmCkTdmCdGdAdAdGfTd TACTCGAAGTGGCTGC 46 72 15 GdGdmCdTkGkmCk 664873 157 172 TkAkmCkTdmCdGdAdAdGdUf TACTCGAAGUGGCTGC 42 72 63 GdGdmCdTkGkmCk 664874 157 172 TkAkmCkTdmCdGdAdAdGdTd TACTCGAAGTGGCTGC 48 69 15 GfGdmCdTkGkmCk 664875 157 172 TkAkmCkTdmCdGdAdAdGdTd TACTCGAAGTGGCTGC 44 66 15 GdGfmCdTkGkmCk 664876 157 172 TkAkmCkTdmCdGdAdAdGdTd TACTCGAAGTGGCTGC 69 77 15 GdGdCfTkGkmCk 664877 150 164 GkTkGdGdmCdTdGdmCdGdTd GTGGCTGCGTACCAC  9 43 22 AdmCdmCkAemCk 664878 151 165 AkGkTdGdGdmCdTdGdmCdG AGTGGCTGCGTACCA 45 82 23 dTdAdmCkmCeAk 664879 152 166 AkAkGdTdGdGdmCdTdGdmC AAGTGGCTGCGTACC 41 72 24 dGdTdAkmCemCk 664880 153 167 GkAkAdGdTdGdGdmCdTdGd GAAGTGGCTGCGTAC 29 58 25 mCdGdTkAemCk 664881 154 168 mCkGkAdAdGdTdGdGdmCdT CGAAGTGGCTGCGTA 35 63 26 dGdmCdGkTeAk 664882 155 169 TkmCkGdAdAdGdTdGdGdmC TCGAAGTGGCTGCGT 40 63 27 dTdGdmCkGeTk 664883 156 170 mCkTkmCdGdAdAdGdTdGdG CTCGAAGTGGCTGCG 21 67 28 cdCdTdGkmCeGk 664884 157 171 AkmCkTdmCdGdAdAdGdTdG ACTCGAAGTGGCTGC 53 78 29 dGdmCdTkGcmCk 664885 158 172 TkAkmCdTdmCdGdAdAdGdTd TACTCGAAGTGGCTG 49 78 30 GdGdmCkTeGk 664886 159 173 GkTkAdmCdTdmCdGdAdAdG GTACTCGAAGTGGCT 51 64 31 dTdGdGkmCeTk 664887 160 174 GkGkTdAdmCdTdmCdGdAdA GGTACTCGAAGTGGC 64 76 32 dGdTdGkGemCk 664899 150 164 GkTkGkGdmCdTdGdmCdGdTd GTGGCTGCGTACCAC 0 13 22 AdmCdmCkAkmCk 664900 151 165 AkGkTkGdGdmCdTdGdmCdG AGTGGCTGCGTACCA 52 81 23 dTdAdmCkmCkAk 664901 152 166 AkAkGkTdGdGdmCdTdGdmC AAGTGGCTGCGTACC 52 84 24 dGdTdAkmCkmCk 664902 153 167 GkAkAkGdTdGdGdmCdTdGd GAAGTGGCTGCGTAC 41 77 25 mCdGdTkAkmCk 664903 154 168 mCkGkAkAdGdTdGdGdmCdT CGAAGTGGCTGCGTA 64 80 26 dGdmCdGkTkAk 664904 155 169 TkmCkGkAdAdGdTdGdGdmC TCGAAGTGGCTGCGT 43 45 27 dTdGdmCkGkTk 664905 156 170 mCkTkmCkGdAdAdGdTdGdG CTCGAAGTGGCTGCG 48 68 28 dmCdTdGkmCkGk 664906 157 171 AkmCkTkmCdGdAdAdGdTdG ACTCGAAGTGGCTGC 59 77 29 dGdmCdTkGkmCk 664907 158 172 TkAkmCkTdmCdGdAdAdGdTd TACTCGAAGTGGCTG 51 71 30 GdGdmCkTkGk 664908 159 173 GkTkAkmCdTdmCdGdAdAdG GTACTCGAAGTGGCT 55 67 31 dTdGdGkmCkTk 664909 160 174 GkGkTkAdmCdTdmCdGdAdA GGTACTCGAAGTGGC 65 69 32 dGdTdGkGkmCk

Example 4: Potency and Selectivity of Antisense Oligonucleotides Targeting the Mutant P23H Rhodopsin Gene

Antisense oligonucleotides from Example 3 exhibiting potent in vitro inhibition of the mutant P23H rhodopsin mRNA were selected and tested at various doses in HEK-293 cells transfected with either the mutant P23H (E5-M) or wild-type (E5-C) rhodopsin/SOD1 minigene construct.

The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cells were transfected using electroporation with 1.25 μM, 2.50 μM, 5.00 μM, 10.00 μM, and 20 μM concentrations of antisense oligonucleotide, as specified in the Tables below. After a treatment period of approximately 16 hours, RNA was isolated from the cells and rhodopsin mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS4220 was used to measure mRNA levels. Rhodopsin mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of rhodopsin, relative to untreated control cells.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented. Several antisense oligonucleotides selectively inhibited expression of the mutant P23H rhodopsin sequence compared to the WT sequence.

TABLE 6 Percent inhibition of wild-type rhodopsin mRNA in WT HEK293 cells (E5-C) ISIS No 1.250 μM 2.50 μM 5.00 μM 10.00 μM 20.00 μM IC₅₀ (μM) 598206 2 20 34 44 58 13 664833 0 8 0 24 18 >20 664836 0 13 7 29 39 >20 664843 0 2 14 20 13 >20 664844 0 2 12 16 6 >20 664846 0 8 14 33 52 19 664849 0 0 4 0 5 >20 664860 0 0 0 0 3 >20 664867 0 12 8 29 33 >20 664876 2 1 20 17 41 >20 664887 0 0 14 14 0 >20 664903 0 0 2 9 0 >20 664906 5 2 35 19 44 >20 664909 0 6 9 4 4 >20

TABLE 7 Percent inhibition of P23H rhodopsin mRNA in mutant HEK293 cells (E5-M) 1.250 2.50 5.00 10.00 20.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 598206 24 45 56 74 83 4 664833 11 37 49 60 66 6 664836 8 37 40 58 70 8 664843 40 42 48 62 61 5 664844 36 50 51 65 59 3 664846 0 17 31 45 63 12 664849 21 41 58 49 60 9 664860 21 43 54 60 72 4 664867 40 47 52 61 69 3 664876 2 27 58 67 67 4 664887 49 51 60 66 68 2 664903 40 48 58 72 73 3 664906 32 46 47 61 67 5 664909 28 47 58 60 54 3

TABLE 8 Percent inhibition of wild-type rhodopsin mRNA in WT HEK293 cells (E5-C) ISIS No 1.250 μM 2.50 μM 5.00 μM 10.00 μM 20.00 μM IC₅₀ (μM) 598206 0 15 31 51 60 10 664824 1 12 25 38 47 >20 664835 0 2 13 24 52 19 664838 0 2 0 23 26 >20 664840 8 13 23 22 40 >20 664848 0 0 10 6 14 >20 664858 9 22 21 48 51 17 664878 5 1 20 33 60 16 664884 6 10 19 30 50 >20 664885 0 0 0 22 0 >20 664900 16 28 31 45 55 15 664901 13 11 26 45 56 14 664902 0 3 0 22 19 >20 664908 0 15 4 18 14 >20

TABLE 9 Percent inhibition of P23H rhodopsin mRNA in mutant HEK293 cells (E5-M) 1.250 2.50 5.00 10.00 20.00 IC₅₀ ISIS No μM μM μM μM μM (μM) 598206 30 44 58 72 84 3 664824 21 36 45 59 62 7 664835 1 16 29 36 66 11 664838 6 27 33 47 63 11 664840 3 45 29 35 62 14 664848 10 16 35 51 59 11 664858 55 58 53 62 70 4 664878 6 32 47 51 72 7 664884 28 37 51 57 68 6 664885 6 10 20 51 69 11 664900 44 51 52 65 71 2 664901 42 50 53 68 70 3 664902 0 27 38 57 64 8 664908 30 45 49 57 58 6

Example 5: Characterization of Potency and Selectivity of Human Antisense Compounds Targeting Mutant P23H Rhodopsin

Several additional antisense oligonucleotides were designed to target the mutant P23H rhodopsin gene and were transfected into either mutant P23H rhodopsin (E5-M) or wild-type (E5-C) rhodopsin/SOD1 minigene HEK293 cells. The SOD1 minigene sequence contains the unspliced sequence of SOD1 exon 4, intron 4, and exon 5, with the human wild-type rhodopsin or a rhodopsin sequence with the mutation at P23H. Each sequence was cloned into pcDNA4/TO at HindIII/EcoRI site.

Study 1

The newly designed chimeric antisense oligonucleotides in the Table below were designed as deoxy, MOE and cEt oligonucleotides with a 7 or 8 base deoxy gap Antisense oligonucleotides having a 7 or 8 base deoxy gap are potent and selective for targeting the SNP mutation of the hungtingtin (HTT) gene. Ostergaard M E et al., Nucleic Acids Res. 2013 November; 41(21):9634-50; PCT Publication WO 2013/022990. It was expected that antisense oligonucleotides having a 7 or 8 base deoxy gap likewise would potently and selectively target P23H rhodopsin.

The ‘Chemistry’ column of the Table below presents chemical modifications in the oligonucleotide, including the position of the sugar modifications, wherein ‘e’ indicates a MOE modification, ‘k’ indicates a cEt modification, and the number indicates the number of deoxyribose sugars. All the oligonucleotides are 16 nucleosides in length. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosines are 5-methylcytosines. “Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. The antisense oligonucleotides were designed to target the human mutant P23H rhodopsin sequence (SEQ ID NO:2). The oligonucleotides are presented in the Table below.

TABLE 10 Antisense oligonucleotides targeting mutant P23H rhodopsin (SEQ ID NO: 2) SEQ ID SEQ ID NO: 2 NO: 2 ISIS Start Stop SEQ ID No Site Site Sequence Chemistry NO 589177 148 163 TGGCTGCGTACCACAC cckk-8-kkcc 33 589193 148 163 TGGCTGCGTACCACAC eeekk-7-kkee 33 589178 149 164 GTGGCTGCGTACCACA eekk-8-kkee 13 589194 149 164 GTGGCTGCGTACCACA eeekk-7-kkee 13 589179 150 165 AGTGGCTGCGTACCAC eekk-8-kkee 16 589195 150 165 AGTGGCTGCGTACCAC eeekk-7-kkee 16 589180 151 166 AAGTGGCTGCGTACCA eekk-8-kkee 11 589196 151 166 AAGTGGCTGCGTACCA eeekk-7-kkee 11 589181 152 167 GAAGTGGCTGCGTACC eekk-8-kkee 17 589197 152 167 GAAGTGGCTGCGTACC eeekk-7-kkee 17 589182 153 168 CGAAGTGGCTGCGTAC eekk-8-kkee 14 589198 153 168 CGAAGTGGCTGCGTAC eeekk-7-kkee 14 589183 154 169 TCGAAGTGGCTGCGTA eekk-8-kkee 18 589199 154 169 TCGAAGTGGCTGCGTA eeekk-7kkee 18 589184 155 170 CTCGAAGTGGCTGCGT eekk-8-kkee 12 589200 155 170 CTCGAAGTGGCTGCGT eeekk-7-kkee 12 589185 156 171 ACTCGAAGTGGCTGCG eekk-8-kkee 19 589201 156 171 ACTCGAAGTGGCTGCG eeekk-7-kkee 19 589186 157 172 TACTCGAAGTGGCTGC eekk-8-kkee 15 589202 157 172 TACTCGAAGTGGCTGC eeekk-7-kkee 15 589187 158 173 GTACTCGAAGTGGCTG eekk-8-kkee 20 589203 158 173 GTACTCGAAGTGGCTG eeekk-7-kkee 20 589188 159 174 GGTACTCGAAGTGGCT eekk-8-kkee 21 589204 159 174 GGTACTCGAAGTGGCT eeekk-7-kkee 21 589189 160 175 GGGTACTCGAAGTGGC eekk-8-kkee 34 589205 160 175 GGGTACTCGAAGTGGC eeekk-7-kkee 34 589190 161 176 TGGGTACTCGAAGTGG eekk-8-kkee 35 589206 161 176 TGGGTACTCGAAGTGG eeekk-7-kkee 35 589191 162 177 GTGGGTACTCGAAGTG eekk-8-kkee 36 589207 162 177 GTGGGTACTCGAAGTG eeekk-7-kkee 36 589192 163 178 TGTGGGTACTCGAAGT eekk-8-kkee 37 589208 163 178 TGTGGGTACTCGAAGT eeekk-7-kkee 37

The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. ISIS 564387 and ISIS 598206, described in the studies above, were also included in these assays. Cultured cells at a density of 20,000 cells per well were transfected using electroporation with antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and rhodopsin mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS4220 was used to measure mRNA levels. Rhodopsin mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of rhodopsin, relative to untreated control cells. A zero value only indicates that the antisense oligonucleotide did not inhibit mRNA expression.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented. Some antisense oligonuclotides selectively reduced mutant P23H rhodopsin mRNA levels compared to WT rhodopsin expression.

TABLE 11 Percent reduction of mutant P23H rhodopsin mRNA in mutant HEK293 cells (E5-M) 740.7 2222.2 6666.7 20000.0 IC₅₀ ISIS No nM nM nM nM (μM) 564387 34 48 70 83 2 589177 22 29 47 65 8 589178 18 8 7 27 >20 589179 10 16 16 33 >20 589180 21 35 56 73 5 589181 20 22 50 67 8 589182 31 40 59 72 4 589183 17 44 47 64 7 589184 27 25 40 60 11 589185 1 30 37 61 11 589186 21 34 40 62 10 589187 28 37 59 64 5 589188 23 25 53 65 8 589189 16 19 48 56 11 589190 20 36 50 64 7 589191 0 20 40 49 17 589192 9 22 39 54 15 598206 41 54 72 84 1

TABLE 12 Percent reduction of wild-type rhodopsin mRNA in WT HEK293 cells (E5-C) 740.7 2222.2 6666.7 20000.0 IC₅₀ ISIS No nM nM nM nM (μM) 564387 2 24 40 70 9 589177 14 27 31 64 13 589178 0 5 0 24 >20 589179 0 16 4 31 >20 589180 18 19 30 48 >20 589181 0 9 15 33 >20 589182 0 10 12 15 >20 589183 0 14 0 9 >20 589184 5 0 0 16 >20 589185 3 5 6 3 >20 589186 1 15 24 30 >20 589187 13 7 21 28 >20 589188 6 9 12 28 >20 589189 15 5 18 38 >20 589190 8 3 5 32 >20 589191 4 7 14 20 >20 589192 0 0 2 34 >20 598206 26 18 41 59 12

TABLE 13 Percent reduction mutant of mutant P23H rhodopsin mRNA in HEK293 cells (E5-M) 740.7 2222.2 6666.7 20000.0 IC₅₀ ISIS No nM nM nM nM (μM) 564387 0 51 67 82 2 589193 10 12 28 40 >20 589194 0 11 19 14 >20 589195 5 18 20 27 >20 589196 4 20 39 44 >20 589197 16 18 47 44 >20 589198 13 28 38 52 17 589199 12 18 31 36 >20 589200 2 11 32 52 20 589201 18 23 21 42 >20 589202 10 11 20 29 >20 589203 15 22 36 45 >20 589204 24 29 33 52 18 589205 5 19 27 40 >20 589206 6 9 22 39 >20 589207 4 11 25 51 20 589208 0 10 10 23 >20 598206 33 53 73 83 2

TABLE 14 Percent reduction of wild-type rhodopsin mRNA in WT HEK293 cells (E5-C) 740.7 2222.2 6666.7 20000.0 IC₅₀ ISIS No nM nM nM nM (μM) 564387 0 22 40 60 11 589193 0 2 5 38 >20 589194 0 0 8 13 >20 589195 0 4 9 13 >20 589196 12 0 12 30 >20 589197 14 2 13 20 >20 589198 10 0 18 10 >20 589199 2 0 5 0 >20 589200 0 0 0 20 >20 589201 0 0 0 16 >20 589202 0 18 0 7 >20 589203 10 6 22 28 >20 589204 0 1 10 17 >20 589205 4 3 4 11 >20 589206 0 0 3 20 >20 589207 0 0 0 24 >20 589208 2 0 4 14 >20 598206 9 8 37 51 17

The summary table is shown below and indicates that only some, much fewer than expected, antisense oligonucleotides having a 7 or 8 base deoxy gap potently and selectively reduced mutant P23H rhodopsin mRNA levels compared to WT levels. The data show that the 7 or 8 base deoxy gap motif may not always be effective to potently and selectively target a mutation from one gene to another.

TABLE 15 Selectivity of antisense oligonucleotides IC₅₀ IC₅₀ (μM) (μM) in WT in P23H ISIS Rho Rho No cells cells 564387 11 2 589177 13 8 589178 >20 >20 589179 >20 >20 589180 >20 5 589181 >20 8 589182 >20 4 589183 >20 7 589184 >20 11 589185 >20 11 589186 >20 10 589187 >20 5 589188 >20 8 589189 >20 11 589190 >20 7 589191 >20 17 589192 >20 15 589193 >20 >20 589194 >20 >20 589195 >20 >20 589196 >20 >20 589197 >20 >20 589198 >20 17 589199 >20 >20 589200 >20 20 589201 >20 >20 589202 >20 >20 589203 >20 >20 589204 >20 18 589205 >20 >20 589206 >20 >20 589207 >20 20 589208 >20 >20 598206 17 2 Study 2

Antisense oligonucleotides described in the studies above were further tested. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. ISIS 549144 (GGCCAATACGCCGTCA; designated herein as SEQ ID NO: 89), a 3-10-3 cEt gapmer that does not target any known gene, was used as a control. The results for each experiment are presented in separate tables shown below. Cultured HEK293 cells at a density of 30,000 cells per well were transfected using electroporation with antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and rhodopsin mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS4220, which is targeted to the SOD1 mini gene, was used to measure mRNA levels. Rhodopsin mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of rhodopsin, relative to untreated control cells. A zero value only indicates that the antisense oligonucleotide did not inhibit mRNA expression.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented. Several antisense oligonucleotides reduced mutant rhodopsin mRNA levels potently and selectively.

TABLE 16 Percent reduction of wild-type rhodopsin mRNA in WT HEK293 cells (E5-C) ISIS No 1.25 μM 2.5 μM 5 μM 10 μM 20 μM IC₅₀ (μM) 549144 0 7 0 0 0 >20 598206 2 20 34 44 58 13 664833 0 8 0 24 18 >20 664836 0 13 7 29 39 >20 664843 0 2 14 20 13 >20 664844 0 2 12 16 6 >20 664846 0 8 14 33 52 19 664849 0 0 4 0 5 >20 664860 0 0 0 0 3 >20 664867 0 12 8 29 33 >20 664876 2 1 20 17 41 >20 664887 0 0 14 14 0 >20 664903 0 0 2 9 0 >20 664906 5 2 35 19 44 >20 664909 0 6 9 4 4 >20

TABLE 17 Percent reduction of P23H rhodopsin mRNA in mutant HEK293 cells (E5-M) ISIS No 1.25 μM 2.5 μM 5 μM 10 μM 20 μM IC₅₀ (μM) 549144 0 0 0 2 0 >20 598206 24 45 56 74 83 4 664833 11 37 49 60 66 6 664836 8 37 40 58 70 8 664843 40 42 48 62 61 5 664844 36 50 51 65 59 3 664846 0 17 31 45 63 12 664849 21 41 58 49 60 9 664860 21 43 54 60 72 4 664867 40 47 52 61 69 3 664876 2 27 58 67 67 4 664887 49 51 60 66 68 2 664903 40 48 58 72 73 3 664906 32 46 47 61 67 5 664909 28 47 58 60 54 3

TABLE 18 Percent reduction of wild-type rhodopsin mRNA in WT HEK293 cells (E5-C) ISIS No 1.25 μM 2.5 μM 5 μM 10 μM 20 μM IC₅₀ (μM) 549144 0 0 0 0 0 >20 598206 0 15 31 51 60 10 664824 1 12 25 38 47 >20 664835 0 2 13 24 52 19 664838 0 2 0 23 26 >20 664840 8 13 23 22 40 >20 664848 0 0 10 6 14 >20 664858 9 22 21 48 51 17 664878 5 1 20 33 60 16 664884 6 10 19 30 50 >20 664885 0 0 0 22 0 >20 664900 16 28 31 45 55 15 664901 13 11 26 45 56 14 664902 0 3 0 22 19 >20 664908 0 15 4 18 14 >20

TABLE 19 Percent reduction of P23H rhodopsin mRNA in mutant HEK293 cells (E5-M) ISIS No 1.25 μM 2.5 μM 5 μM 10 μM 20 μM IC₅₀ (μM) 549144 0 0 0 0 0 >20 598206 30 44 58 72 84 3 664824 21 36 45 59 62 7 664835 1 16 29 36 66 11 664838 6 27 33 47 63 11 664840 3 45 29 35 62 14 664848 10 16 35 51 59 11 664858 55 58 53 62 70 4 664878 6 32 47 51 72 7 664884 28 37 51 57 68 6 664885 6 10 20 51 69 11 664900 44 51 52 65 71 2 664901 42 50 53 68 70 3 664902 0 27 38 57 64 8 664908 30 45 49 57 58 6

The summary table is shown below and indicates that some antisense oligonucleotides, including ISIS 664844, potently and selectively reduced mutant rhodopsin mRNA levels compared to WT rhodopsin levels.

TABLE 20 Selectivity of antisense oligonucleotides IC₅₀ IC₅₀ (μM) (μM) in WT in P23H ISIS Rho Rho No cells cells 549144 >20 >20 598206 10 3 664824 >20 7 664833 >20 6 664835 19 11 664836 >20 8 664838 >20 11 664840 >20 14 664843 >20 5 664844 >20 3 664846 19 12 664848 >20 11 664849 >20 9 664858 17 4 664860 >20 4 664867 >20 3 664876 >20 4 664878 16 7 664884 >20 6 664885 >20 11 664887 >20 2 664900 15 2 664901 14 3 664902 >20 8 664903 >20 3 664906 >20 5 664908 >20 6 664909 >20 3 Study 3

Antisense oligonucleotides from the studies described above were further tested. Two new oligonucleotides were designed and are presented in the Table below.

ISIS 586139 is a 3-10-3 cEt gapmer, wherein the central gap segment comprises ten 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising three nucleosides each. ISIS 643801 is a 2-10-2 cEt gapmer, wherein the central gap segment comprises ten 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising two nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a cEt modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. “Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. The antisense oligonucleotides were designed to target the mutant sequence (SEQ ID NO:2). The oligonucleotides are presented in the Table below.

TABLE 21 Antisense oligonucleotides targeting P23H rhodopsin (SEQ ID NO: 2) SEQ ID SEQ ID NO: 2 NO: 2 Start Stop ISIS No Site Site Sequence SEQ ID NO 586139 158 173 GTACTCGAAGTGGCTG 20 643801 152 165 AGTGGCTGCGTACC 38

The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. ISIS 549144 was used as a control. The results for each experiment are presented in separate tables shown below. Cultured HEK293 cells having the SOD-1 minigene at a density of 30,000 cells per well were transfected using electroporation with antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and rhodopsin mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS4220, which is targeted to the SOD1 mini gene, was used to measure mRNA levels. Rhodopsin mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of rhodopsin, relative to untreated control cells. A zero value only indicates that the antisense oligonucleotide did not inhibit mRNA expression.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented. Several antisense oligonucleotides reduced mutant rhodopsin mRNA levels potently and selectively.

TABLE 22 Percent reduction of wild-type rhodopsin mRNA in WT HEK293 cells (E5-C) ISIS No 0.5 μM 1.5 μM 4.4 μM 13.3 μM 40 μM IC₅₀ (μM) 549144 0 1 0 4 0 >40 564387 0 22 42 59 78 8 564389 0 0 0 33 38 >40 564425 2 0 5 17 7 >40 564426 10 19 35 45 61 17 564431 0 0 0 0 4 >40 586139 3 20 15 35 53 33 589177 37 54 53 62 64 3 643801 0 12 27 53 68 14 664838 0 0 0 12 16 >40 664843 0 25 13 41 50 34 664844 0 3 6 10 17 >40 664860 0 0 0 9 10 >40 664867 0 16 4 44 52 29 664884 3 0 0 43 53 28 664885 0 0 0 3 13 >40

TABLE 23 Percent reduction of P23H rhodopsin mRNA in mutant HEK293 cells (E5-M) ISIS No 0.5 μM 1.5 μM 4.4 μM 13.3 μM 40 μM IC₅₀ (μM) 549144 0 0 4 0 10 >40 564387 8 36 69 85 90 3 564389 0 37 64 77 77 3 564425 25 41 47 75 80 3 564426 26 43 49 79 80 2 564431 0 0 0 10 17 >40 586139 34 42 63 75 83 2 589177 28 33 40 60 64 8 643801 1 27 49 72 88 5 664838 0 16 39 45 78 9 664843 23 31 64 76 78 3 664844 29 56 66 75 73 2 664860 11 36 65 77 83 3 664867 17 44 64 76 82 3 664884 0 28 54 71 84 5 664885 0 25 53 73 83 5

The results of studies in mutant and WT cells are summarized in the Table below. The IC₅₀ values show the potency of certain oligonucleotides. The data shows that some oligonucleotides, including ISIS 664844, demonstrate potency and selectivity for the human mutant P23H rhodopsin gene. The sequence of the oligonucleotide with the mutation bolded and underlined is also shown.

TABLE 24 IC₅₀ for the WT and P23H mutant cells ISIS NO Sequence with mutation WT Mutant SEQ ID NO: 564389 CTCGAAG T GGCTGCGT >40 3 12 564426 TACTCGAAG T GGCTGC 17 2 15 664844 GGTACTCGAAG T GGCT >40 2 21 664860 CGAAG T GGCTGCGTAC >40 3 14 664867 TACUCGAAG T GGCTGC 29 3 64 664884 ACTCGAAG T GGCTGC 28 5 29

Example 6: Efficacy of Antisense Oligonucleotides Targeting Human Rhodopsin in Transgenic Mice

Additional antisense oligonucleotides were designed and tested in two transgenic (Tg) mice models. The germline of these mice were inserted with a P23H mutant allele from a retinitis pigmentosa patient (Olsson, J. E. et al., Neuron. 1992. 9: 815-830). A total of 144 antisense oligonucleotides were tested. Not all the antisense oligonucleotides tested demonstrated potency in inhibiting mutant rhodopsin expression.

Study 1

P23HTg mice were treated with ISIS oligonucleotides described in the studies above. Two newly designed 3-10-3 cEt gapmers targeted to rhodopsin away from the P23H site, ISIS 564426 and ISIS 564432, were also included in the study.

TABLE 25 3-10-3 cEt gapmers targeting human rhodopsin (SEQ ID NO: 1) SEQ ID SEQ ID NO: 1 NO: 1 ISIS Start Stop NO Site Site Sequence SEQ ID NO 564429 7798 7813 TAAGAAATGGACCCTA 39 564432 8692 8707 CCCGGGTCCAGACCAT 40

P23H Tg mice were randomly divided into treatment groups of 3-5 mice each. The gapmers were injected at a dose of 50 μg via intravitreal injection in the right eye of each of the mice. The left eye of the animals was injected with PBS and served as the control. Mice were sacrificed after 7 days. Human P23H rhodopsin expression from eye tissue was measured with the human-specific primer probe set RTS3363. The results are normalized to the expression of mouse cone rod homeobox. Percent inhibition is relative to the expression seen in mice treated with PBS. The data are presented in the Table below and demonstrate that some antisense oligonucleotides reduced mutant human P23H rhodopsin expression in vivo.

TABLE 26 % inhibition of human P23H rhodopsin mRNA ISIS No % 564431 89 564299 41 564329 38 564363 21 564370 34 564372 15 564373 33 564422 43 564429 31 564432 6 564433 7 Study 2

The newly designed chimeric antisense oligonucleotides in the Tables below were designed as 3-10-3 cEt gapmers, 5-7-4 cEt gapmers, 5-10-5 MOE gapmers, 6-8-6 MOE gapmers, 7-6-7 MOE gapmers, 4-10-4 MOE gapmers, 5-8-5 MOE gapmers, 4-8-4 MOE gapmers, or 5-6-5 MOE gapmers.

The 3-10-3 cEt gapmers are 16 nucleosides in length, wherein the central gap segment comprises ten 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising three nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a cEt modification. The 5-7-4 cEt gapmers are 16 nucleosides in length, wherein the central gap segment comprises seven 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five and four nucleosides respectively. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a cEt modification. The 5-10-5 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises ten 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a MOE modification. The 6-8-6 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises eight 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising six nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a MOE modification. The 7-6-7 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises six 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising seven nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a MOE modification. The 4-10-4 MOE gapmers are 18 nucleosides in length, wherein the central gap segment comprises ten 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising four nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a MOE modification. The 5-8-5 MOE gapmers are 18 nucleosides in length, wherein the central gap segment comprises eight 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a MOE modification. The 4-8-4 MOE gapmers are 16 nucleosides in length, wherein the central gap segment comprises eight 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising four nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a MOE modification. The 5-6-5 MOE gapmers are 16 nucleosides in length, wherein the central gap segment comprises six 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a MOE modification.

The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines. “Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. The antisense oligonucleotides were designed to target the mutant sequence (SEQ ID NO:2).

P23H Tg mice were randomly divided into treatment groups of 3-5 mice each. The gapmers were injected at a dose of 50 μg via intravitreal injection in the right eye of each of the mice. The left eye of the animals was injected with PBS and served as the control. Mice were sacrificed after 7 days. Human rhodopsin expression was measured with the human-specific primer probe set RTS3363. The results are normalized to the expression of mouse cone rod homeobox. Percent inhibition is relative to the expression seen in mice treated with PBS. A ‘0’ value inhibition only indicates that the oligonucleotide did not inhibit expression of in this particular instance. The data are presented in the Table below.

TABLE 27 Inhibition of rhodopsin expression in P23H Tg mice SEQ ID SEQ ID NO: 2 NO: 2 Start Stop % SEQ ID ISIS NO Site Site Motif Sequence inhibition NO 564426 157 172 3-10-3 cEt TACTCGAAGTGGCTGC 41 15 598213 152 167 5-7-4 cEt GAAGTGGCTGCGTACC 0 17 614060 150 169 5-10-5 MOE TCGAAGTGGCTGCGTACCAC 6 41 614067 157 176 5-10-5 MOE TGGGTACTCGAAGTGGCTGC 0 42 614068 158 177 5-10-5 MOE GTGGGTACTCGAAGTGGCTG 0 43 614074 164 183 5-10-5 MOE AGTACTGTGGGTACTCGAAG 0 44 614075 143 162 6-8-6 MOE GGCTGCGTACCACACCCGTC 9 45 614082 150 169 6-8-6 MOE TCGAAGTGGCTGCGTACCAC 7 41 614083 151 170 6-8-6 MOE CTCGAAGTGGCTGCGTACCA 0 46 614089 157 176 6-8-6 MOE TGGGTACTCGAAGTGGCTGC 0 42 614105 151 170 7-6-7 MOE CTCGAAGTGGCTGCGTACCA 0 46 614111 157 176 7-6-7 MOE TGGGTACTCGAAGTGGCTGC 0 42 614166 150 167 4-10-4 MOE GAAGTGGCTGCGTACCAC 0 47 614167 151 168 4-10-4 MOE CGAAGTGGCTGCGTACCA 34 48 614187 151 168 5-8-5 MOE CGAAGTGGCTGCGTACCA 0 48 614188 152 169 5-8-5 MOE TCGAAGTGGCTGCGTACC 1 49 614194 158 175 5-8-5 MOE GGGTACTCGAAGTGGCTG 0 50 614195 159 176 5-8-5 MOE TGGGTACTCGAAGTGGCT 11 51 614250 158 173 4-8-4 MOE GTACTCGAAGTGGCTG 5 20 614251 159 174 4-8-4 MOE GGTACTCGAAGTGGCT 0 21 614263 153 168 5-6-5 MOE CGAAGTGGCTGCGTAC 0 14 614268 158 173 5-6-5 MOE GTACTCGAAGTGGCTG 16 20 Study 3

The newly designed chimeric antisense oligonucleotides in the Tables below were designed as 3-10-3 cEt gapmers, 6-8-6 MOE gapmers, 4-10-4 MOE gapmers, 5-8-5 MOE gapmers, 6-6-6 MOE gapmers, 3-10-3 MOE gapmers, or 4-8-4 MOE gapmers.

The 3-10-3 cEt gapmers are 16 nucleosides in length, wherein the central gap segment comprises ten 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising three nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a cEt modification. The 6-8-6 MOE gapmers are 20 nucleosides in length, wherein the central gap segment comprises eight 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising six nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a MOE modification. The 4-10-4 MOE gapmers are 18 nucleosides in length, wherein the central gap segment comprises ten 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising four nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a MOE modification. The 5-8-5 MOE gapmers are 18 nucleosides in length, wherein the central gap segment comprises eight 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a MOE modification. The 6-6-6 MOE gapmers are 18 nucleosides in length, wherein the central gap segment comprises six 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising six nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a MOE modification. The 3-10-3 MOE gapmers are 16 nucleosides in length, wherein the central gap segment comprises ten 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising three nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a MOE modification. The 4-8-4 MOE gapmers are 16 nucleosides in length, wherein the central gap segment comprises eight 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising four nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P-S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines.

“Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. The antisense oligonucleotides were designed to target the mutant sequence (SEQ ID NO:2).

P23H Tg mice were randomly divided into treatment groups of 3-5 mice each. The gapmers were injected at a dose of 50 μg via intravitreal injection in the right eye of each of the mice. The left eye of the animals was injected with PBS and served as the control. Mice were sacrificed after 7 days. Human rhodopsin expression was measured with the human-specific primer probe set RTS3363. The results are normalized to the expression of mouse cone rod homeobox. Percent inhibition is relative to the expression seen in mice treated with PBS. A ‘0’ value inhibition only indicates that the oligonucleotide did not inhibit expression of in this particular instance. The data are presented in the Table below.

TABLE 28 Inhibition of rhodopsin expression in P23H Tg mice SEQ ID SEQ ID NO: 2 NO: 2 Start Stop % SEQ ID ISIS NO Site Site Motif Sequence inhibition NO 614225 151 166 3-10-3 MOE AAGTGGCTGCGTACCA 1 11 614208 152 169 6-6-6 MOE TCGAAGTGGCTGCGTACC 0 49 614226 152 167 3-10-3 MOE GAAGTGGCTGCGTACC 3 17 614244 152 167 4-8-4 MOE GAAGTGGCTGCGTACC 2 17 614227 153 168 3-10-3 MOE CGAAGTGGCTGCGTAC 0 14 614245 153 168 4-8-4 MOE CGAAGTGGCTGCGTAC 0 14 614246 154 169 4-8-4 MOE TCGAAGTGGCTGCGTA 0 18 614088 156 175 6-8-6 MOE GGGTACTCGAAGTGGCTGCG 3 52 614192 156 173 5-8-5 MOE GTACTCGAAGTGGCTGCG 5 53 614193 157 174 5-8-5 MOE GGTACTCGAAGTGGCTGC 6 54 614231 157 172 3-10-3 MOE TACTCGAAGTGGCTGC 11 15 614232 158 173 3-10-3 MOE GTACTCGAAGTGGCTG 10 20 614233 159 174 3-10-3 MOE GGTACTCGAAGTGGCT 0 44 586141 160 175 3-10-3 cEt GGGTACTCGAAGTGGC 0 34 586143 162 177 3-10-3 cEt GTGGGTACTCGAAGTG 0 36 614178 162 179 4-10-4 MOE CTGTGGGTACTCGAAGTG 30 55 564340 1133 1148 3-10-3 cEt CAGGTCTTAGGCCGGG 20 56 Study 4

The newly designed chimeric antisense oligonucleotides in the Tables below were designed as 3-10-3 cEt gapmers or deoxy, 2′-fluoro and cEt oligonucleotides. The 3-10-3 cEt gapmers are 16 nucleosides in length, wherein the central gap segment comprises ten 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising three nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a cEt modification. The deoxy, 2′-fluoro and cEt oligonucleotides are 16 nucleosides in length. The ‘Chemistry’ column of the Table below presents the position of the sugar modifications, wherein ‘e’ indicates a MOE modification, ‘k’ indicates a cEt modification, d indicates a deoxyribose sugar, and ‘f’ indicates a 2′-alpha-fluoro modification; ‘mC’ indicates 5-methycytosine; ‘A’, ‘C’, ‘T’, ‘G’, and ‘U’ represent the standard nucleotide notations The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages.

“Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. The gapmers are targeted to either the human rhodopsin genomic sequence, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NT 005612.16 truncated from nucleotides 35737800 to 35755500) or the mutant sequence (SEQ ID NO:2), or both. ‘n/a’ indicates that the antisense oligonucleotide does not target that particular gene sequence with 100% complementarity.

P23H Tg mice were randomly divided into treatment groups of 3-5 mice each. ISIS 564340 from the studies described above was also included in this assay. 3-10-3 cEt gapmers were injected at a dose of 50 μg via intravitreal injection in the right eye of each of the mice. The left eye of the animals was injected with PBS and served as the control. Mice were sacrificed after 7 days. Human rhodopsin expression from eye tissue was measured with the human-specific primer probe set RTS3363. The results are normalized to the expression of mouse cone rod homeobox. Percent inhibition is relative to the expression seen in mice treated with PBS.

TABLE 29 Inhibition of rhodopsin expression in P23H Tg mice SEQ ID SEQ ID % NO: 2 NO: 2 SEQ ISIS inhibi- Start Stop ID NO Motif Sequence tion Site Site NO 586138 AkmCkTkmCdGdAdAdGdTdGdGdmCdTdGkmCkGk ACTCGAAGTGGCTGCG 24  156  171 19 598204 AkAkGkTdGdGfmCdTdGdmCdGdTdAdmCkmCkAk AAGTGGCTGCGTACCA  2  151  166 11 598208 AkAkGkTdGdGdmCdTdGdCfGdTdAdmCkmCkAk AAGTGGCTGCGTACCA 15  151  166 11 598211 AkAkGkTdGdGdmCdTdGdmCdGdTdAfmCkmCkAk AAGTGGCTGCGTACCA 13  151  166 11 564340 mCkAkGkGdTdmCdTdTdAdGdGdmCdmCdGkGkGk CAGGTCTTAGGCCGGG 21 1133 1148 56 Study 5

Additional oligonucleotides were designed with the same sequence as antisense oligonucleotides described above but with different chemistries. The newly designed chimeric antisense oligonucleotides in the Tables below were designed as 3-10-3 cEt gapmers or deoxy, 2′-fluoro and cEt oligonucleotides. The 3-10-3 cEt gapmers are 16 nucleosides in length, wherein the central gap segment comprises ten 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising three nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a cEt modification. The deoxy, 2′-fluoro and cEt oligonucleotides are 16 nucleosides in length. The ‘Chemistry’ column of the Table below presents the position of the sugar modifications, wherein ‘e’ indicates a MOE modification, ‘k’ indicates a cEt modification, d indicates a deoxyribose sugar, and ‘f’ indicates a 2′-alpha-fluoro modification; ‘mC’ indicates 5-methycytosine; ‘A’, ‘C’, ‘T’, ‘G’, and ‘U’ represent the standard nucleotide notations. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages.

‘Parent oligo’ indicates the ISIS oligonucleotide with the same sequence as the newly designed oligonucleotide. “Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. The gapmers are targeted to the human mutant P23H sequence (SEQ ID NO:2). ‘n/a’ indicates that the antisense oligonucleotide does not target that particular gene sequence with 100% complementarity.

TABLE 30 Antisense oligonucleotides targeting SEQ ID NO: 2 SEQ ID SEQ ID NO: 2 NO: 2 SEQ ISIS Parent Start Stop ID NO oligo Site Site Motif Sequence NO 586136 — 152 167 GkAkAkGdTdGdGdmCdTdGdmCdGdTdAkmCkmCk GAAGTGGCTGCGTACC 17 586137 — 154 169 TkmCkGkAdAdGdTdGdGdmCdTdGdmCdGkTkAk TCGAAGTGGCTGCGTA 18 598212 586136 152 167 GkAkAkGkTdGdGdmCdTdGdmCdGdTkAkmCkmCk GAAGTGGCTGCGTACC 17 598214 561125 153 168 mCkGkAkAkGdTdGdGdmCdTdGdmCdGkTkAkmCk CGAAGTGGCTGCGTAC 14 598215 564425 153 168 mCkGkAkAkGkTdGdGdmCdTdGdmCdGkTkAkmCk CGAAGTGGCTGCGTAC 14 598216 586137 154 169 TkmCkGkAkAdGdTdGdGdmCdTdGdmCkGkTkAk TCGAAGTGGCTGCGTA 18 598217 586137 154 169 TkmCkGkAkAkGdTdGdGdmCdTdGdmCkGkTkAk TCGAAGTGGCTGCGTA 18 598218 564389 155 170 mCkTkmCkGkAdAdGdTdGdGdmCdTdGkmCkGdTk CTCGAAGTGGCTGCGT 12 598219 564389 155 170 mCkTkmCkGkAkAdGdTdGdGdmCdTdGkmCkGkTk CTCGAAGTGGCTGCGT 12

P23H Tg mice were randomly divided into treatment groups of 3-5 mice each. ISIS 564431 and ISIS 598206, described in the studies above were also included in this assay. The antisense oligonucleotides were injected at a dose of 50 μg via intravitreal injection in the right eye of each of the mice. The left eye of the animals was injected with PBS and served as the control. Mice were sacrificed after 7 days. Human rhodopsin expression from eye tissue was measured with the human-specific primer probe set RTS3363. The results are normalized to the expression of mouse cone rod homeobox. Percent inhibition is relative to the expression seen in mice treated with PBS. A ‘0’ value inhibition only indicates that the oligonucleotide did not inhibit expression of in this particular instance. The data are presented in the Table below and demonstrate that some antisense oligonucleotides reduced mutant human rhodopsin expression in vivo.

TABLE 31 Percent inhibition of mutant P23H rhodopsin expression ISIS NO % inhibition 564431 64 586136 29 586137 19 598206 51 598209 14 598210 10 598212 8 598214 47 598215 10 598216 25 598217 4 598218 20 598219 10

Example 7: Potency and Selectivity of Human Antisense Compounds Targeting Human Mutant P23H Rhodopsin

Additional antisense oligonucleotides were designed targeting the P23H site of human mutant P23H rhodopsin. These oligonucleotides as well as antisense oligonucleotides described in the studies above were further tested. The oligonucleotides were transfected into either HEK293 cells expressing either P23H mutant rhodopsin/SOD1 minigene (E5-M) or wild-type rhodopsin/SOD1 minigene (E5-C).

The new antisense oligonucleotides were designed as 3-10-3 cEt gapmers. The gapmers are 16 nucleosides in length, wherein the central gap segment comprises ten 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising three nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a cEt modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages.

“Start site” indicates the 5′-most nucleoside to which the gapmer is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the gapmer is targeted human gene sequence. The gapmers are targeted to the human mutant P23H sequence (SEQ ID NO:2. ‘Mismatch indicates the number of mismatches the oligonucleotide has with the rhodopsin sequence in addition the to P23H mutation

TABLE 32 3-10-3 cEt gapmers targeted to SEQ ID NO: 2 Start Stop Mis- Site Site matches on on with SEQ SEQ ID SEQ ID SEQ ID ID ISIS No Sequence NO: 2 NO: 2 NO: 2 NO 586125 GGGGCTGCGTACCACA 149 164 1 57 586126 AAGGGGCTGCGTACCA 151 166 1 58 586127 CGAAGGGGCTGCGTAC 153 168 1 59 586128 CTCGAAGGGGCTGCGT 155 170 1 60 586129 TACTCGAAGGGGCTGC 157 172 1 61

The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. The results for each experiment are presented in separate tables shown below. Cultured cells at a density of 30,000 cells per well were transfected using electroporation with antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and rhodopsin mRNA levels were measured by quantitative real-time PCR. Human primer probe set RTS3374 was used to measure mRNA levels. Rhodopsin mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of rhodopsin, relative to untreated control cells. A zero value only indicates that the antisense oligonucleotide did not inhibit mRNA expression.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotide is also presented. Several antisense oligonucleotides differentially reduced mutant rhodopsin mRNA levels compared to WT rhodopsin expression.

TABLE 33 Percent reduction of wild-type rhodopsin mRNA in WT HEK293 cells (E5-C) 0.74 2.22 6.67 20.00 IC₅₀ ISIS No μM μM μM μM (μM) 564389 0 25 26 67 12 564425 0 16 31 55 16 586136 6 23 54 72 7 586137 0 18 28 58 15 598202 25 34 60 79 4 598203 10 26 43 69 8 598204 12 30 50 81 5 598205 0 21 39 66 10 598206 23 28 68 81 4 598207 0 15 53 70 8 598208 22 38 64 81 4 598209 0 18 50 75 7 598210 10 14 45 76 8 598211 14 39 69 80 4 598212 19 16 27 45 >20 598213 25 0 30 61 14 564325 17 22 35 53 17 564431 35 36 45 66 7 564387 18 35 53 53 6

TABLE 34 Percent reduction of P23H rhodopsin mRNA in mutant HEK293 cells (E5-M) ISIS 0.74 2.22 6.67 20.00 IC₅₀ No μM μM μM μM (μM) 564389 15 33 42 57 11 564425 0 39 49 58 8 586136 14 33 55 72 6 586137 10 39 26 62 13 598202 20 40 58 65 4 598203 0 20 46 57 11 598204 8 29 52 61 8 598205 1 24 38 59 12 598206 15 49 66 67 3 598207 16 29 49 54 11 598208 20 30 59 54 5 598209 17 33 53 63 7 598210 14 29 50 68 7 598211 17 39 58 77 4 598212 14 21 51 64 8 598213 8 13 27 42 >20 564325 31 18 29 56 17 564431 15 33 45 54 12 564387 24 32 51 51 12

TABLE 35 Percent reduction of wild-type rhodopsin mRNA in WT HEK293 cells (E5-C) ISIS 0.74 2.22 6.67 20.00 IC₅₀ No μM μM μM μM (μM) 598214 0 4 28 51 19 598215 0 9 17 41 >20 598216 0 3 16 48 >20 598217 0 6 10 30 >20 598218 0 8 18 25 >20 598219 0 9 7 29 >20 564389 13 0 36 63 14 564424 10 4 31 47 >20 564425 0 0 20 60 19 564426 0 16 47 56 11 586125 35 49 69 74 2 586126 18 27 57 71 6 586127 12 25 51 68 7 586128 14 37 50 65 7 586129 52 67 81 83 1 564325 25 28 36 61 12 564431 13 41 59 60 4 564387 7 12 54 76 7

TABLE 36 Percent reduction of P23H rhodopsin mRNA in mutant HEK293 cells (E5-M) ISIS 0.74 2.22 6.67 20.00 IC₅₀ No μM μM μM μM (μM) 598214 32 46 57 72 3 598215 21 39 39 66 8 598216 18 26 30 53 17 598217 7 20 16 50 20 598218 5 6 21 48 >20 598219 0 13 33 45 >20 564389 1 39 31 60 12 564424 0 24 25 44 >20 564425 20 41 51 54 9 564426 19 31 50 60 8 586125 0 19 25 53 20 586126 15 22 35 42 >20 586127 7 13 4 28 >20 586128 2 10 18 18 >20 586129 17 19 34 48 >20 564325 30 23 33 50 19 564431 2 24 39 42 >20 564387 11 12 53 64 9

The summary table is shown below and indicates that only a few antisense oligonucleotides selectively reduced mutant rhodopsin mRNA levels compared to WT rhodopsin levels. A selectivity of 1′ indicates that the antisense oligonucleotide did not selectively reduce the mutant sequence compared to the control. A negative selectivity value indicates that the antisense oligonucleotide targeted the wild-type sequence more potently than the mutant sequence.

TABLE 37 Selectivity of antisense oligonucleotides ISIS No Selectivity 598214 5.8 598215 2.6 598216 1.2 598217 1.0 598218 1.0 598219 1.0 564389 1.0 564424 1.0 564425 1.7 564426 1.4 586125 −9.5 586126 −3.5 586127 −2.8 586128 −2.9 586129 −31.5 564325 −1.6 564431 −5.0 564387 −1.2 564389 1.0 564425 2.0 586136 1.2 586137 1.2 598202 1.0 598203 −1.4 598204 −1.5 598205 −1.2 598206 1.3 598207 1.5 598208 −1.3 598209 1.0 598210 1.2 598211 −1.1 598212 2.5 598213 −1.5 564325 1.0 564431 −1.7 564387 −2.1

Example 8: Efficacy and Selectivity of Antisense Oligonucleotides Targeting Human Rhodopsin in Transgenic Mice

Antisense oligonucleotides selected from the studies described above were further tested in transgenic mouse models. The germline of these mice were inserted with either a wild-type rhodopsin allele or a P23H mutant rhodopsin allele from a retinitis pigmentosa patient.

Study 1

P23H Tg mice were randomly divided into treatment groups of 4 mice each. ISIS oligonucleotides were injected via intravitreal injection in the right eye of each of the mice. The left eye of the animals was injected with PBS and served as the control. Mice were sacrificed after 7 days. Human rhodopsin expression from eye tissue was measured with the human-specific primer probe set RTS3363. The results are normalized to the expression of mouse cone rod homeobox. Percent inhibition is relative to the expression seen in the eye tissue treated with PBS. A ‘0’ value inhibition only indicates that the oligonucleotide did not inhibit expression of in this particular instance. The data are presented in the Table below and demonstrated that the antisense oligonucleotides inhibit expression of mutant P23H rhodopsin gene in a dose-dependent manner.

TABLE 38 % inhibition of human mutant P23H rhodopsin expression ISIS No Chemistry Dose (μg) % inhibition 564431 3-10-3 cEt 20 64 564426 3-10-3 cEt 50 63 20 42 10 12 664844 Deoxy, MOE, 50 50 and cEt 20 41 10 32 664860 Deoxy, MOE, 50 44 and cEt 20 39 10 30 664867 Deoxy, 2′-alpha- 50 62 fluoro and cEt 20 25 10 0 664884 Deoxy, MOE, 50 68 and cEt 20 48 10 17 Study 2

Human WT rhodopsin Tg mice were randomly divided into treatment groups of 3-6 mice each. ISIS oligonucleotides, selected from the studies described above, were injected via intravitreal injection in the right eye of each of the mice. The left eye of the animals was injected with PBS and served as the control. Mice were sacrificed after 7 days. Human rhodopsin expression from eye tissue was measured with the human-specific primer probe set RTS3363. The results are normalized to the expression of mouse cone rod homeobox. Percent inhibition is relative to the expression seen in the eye tissue treated with PBS. A ‘0’ value inhibition only indicates that the oligonucleotide did not inhibit expression of in this particular instance. The results are presented in the Table below and demonstrate the several antisense oligonucleotides do not effectively inhibit expression of the wild-type rhodopsin gene.

TABLE 39 % reduction in human WT rhodopsin expression ISIS No Chemistry Dose (μg) % inhibition 564389 3-10-3 cEt 50 10 20 0 10 10 564426 3-10-3 cEt 50 21 20 3 10 0 664844 Deoxy, MOE 50 22 and cEt 20 24 10 0 664860 Deoxy, MOE 50 39 and cEt 20 19 10 5 664884 Deoxy, MOE 50 28 and cEt 20 0 10 2 664867 Deoxy, 2′- 50 9 alpha-fluoro 20 16 and cEt 10 7

Example 9: Confirmation of Efficacy and Selectivity of Antisense Oligonucleotides Targeting Human Rhodopsin in Transgenic Mice

Select antisense oligonucleotides that demonstrated potency and selectivity in the studies described above were further tested in the human P23H or wild-type rhodopsin transgenic mouse models. The data demonstrates the selectivity of the leads for the mutant rhodopsin gene.

Study 1

P23H Tg mice were randomly divided into treatment groups of 4 mice each. ISIS oligonucleotides, selected from the studies described above, were injected via intravitreal injection in the right eye of each of the mice. The left eye of the animals was injected with PBS and served as the control. Mice were sacrificed after 7 days. Human rhodopsin expression from eye tissue was measured with the human-specific primer probe set RTS3363. The results are normalized to the expression of mouse cone rod homeobox. Percent inhibition is relative to the expression seen in the eye treated with PBS. The data presented in the Table below are the average of two separate experiments and demonstrate that the antisense oligonucleotides inhibit expression of mutant rhodopsin gene in a dose-dependent manner.

TABLE 40 % inhibition of human mutant P23H rhodopsin expression ISIS No Chemistry Dose (μg) % inhibition 564426 3-10-3 cEt 50 68 35 45 20 27 664844 Deoxy, MOE, 50 40 and cEt 35 37 20 20 664867 Deoxy, 2′- 50 58 alpha-fluoro 35 43 and cEt 20 26 664884 Deoxy, MOE, 50 51 and cEt 35 48 20 25 Study 2

Human WT rhodopsin Tg mice were randomly divided into treatment groups of 4 mice each. ISIS oligonucleotides, selected from the studies described above, were injected via intravitreal injection in the right eye of each of the mice. The left eye of the animals was injected with PBS and served as the control. Mice were sacrificed after 7 days. Human rhodopsin expression from eye tissue was measured with the human-specific primer probe set RTS3363. The results are normalized to the expression of mouse cone rod homeobox. Percent inhibition is relative to the expression seen in the eye treated with PBS. The data presented in the Table below are the average of two separate experiments and demonstrate that the antisense oligonucleotides do not target the WT rhodopsin gene.

TABLE 41 % inhibition of human WT rhodopsin expression ISIS No Chemistry Dose (μg) % inhibition 564426 3-10-3 cEt 50 13 35 13 664844 Deoxy, MOE, 50 16 and cEt 35 17 664867 Deoxy, 2′- 50 12 alpha-fluoro 35 3 and cEt 664884 Deoxy, MOE, 50 14 and cEt 35 1

Example 10: Tolerability Study of Antisense Oligonucleotides Targeting Human Mutant P23H Rhodopsin in Cynomolgus Monkeys

Cynomolgus monkeys were treated with ISIS antisense oligonucleotides selected from studies described in the Examples above. The objective of this study was to determine the tolerability of the antisense oligonucleotides when given as a single intravitreal injection to cynomolgous monkeys. A cynomolgus surrogate ASO, ISIS 602881, was included in the study.

At the time this study was undertaken, the cynomolgus monkey genomic sequence was not available in the National Center for Biotechnology Information (NCBI) database; therefore, cross-reactivity with the cynomolgus monkey gene sequence could not be confirmed. Instead, the sequences of the ISIS antisense oligonucleotides used in the cynomolgus monkeys was compared to a rhesus monkey sequence for homology. It is expected that ISIS oligonucleotides with homology to the rhesus monkey sequence are fully cross-reactive with the cynomolgus monkey sequence as well. The human antisense oligonucleotides tested are cross-reactive with the rhesus genomic sequence (the complement of GENBANK Accession No. NW_001096632.1 truncated from nucleotides 1522000 to 1532000, designated herein as SEQ ID NO: 4). The greater the complementarity between the human oligonucleotide and the rhesus monkey sequence, the more likely the human oligonucleotide can cross-react with the cynomolgus monkey sequence. “Start site” indicates the 5′-most nucleotide to which the gapmer is targeted in the rhesus monkey gene sequence. ‘Mismatches’ indicates the number of nucleobases mismatched between the human oligonucleotide sequence and the rhesus monkey genomic sequence.

TABLE 42 Antisense oligonucleotides complementary to the rhesus rhodopsin genomic sequence (SEQ ID NO: 4) Target SEQ Start ID ISIS No Site Mismatches Sequence Chemistry NO 564426 1525 1 TACTCGAAGTGGCTGC 3-10-3 cEt 15 664867 1525 1 TACUCGAAGTGGCTGC Deoxy, 2′-alpha-fluoro 64 and cEt 664884 1525 1 ACTCGAAGTGGCTGC Deoxy, MOE and cEt 29 664844 1527 1 GGTACTCGAAGTGGCT Deoxy, MOE and cEt 21 602881 6434 0 TCATTCTGCACAGGCG 3-10-3 cEt 70 Treatment

Prior to the study, the monkeys were kept in quarantine during which the animals were observed daily for general health. The monkeys were 2-4 years old and weighed between 2 and 6 kg. The monkeys were randomized and assigned to groups, as shown the Table below. The monkeys were injected in the left eye (OS) with either PBS or various ASO doses and in the right eye (OD) with various ASO doses. ‘OS’ stands for ‘oculus sinister’ (left cyc) and ‘OD’ stands for ‘oculus dexter’ (right eye).

TABLE 43 Monkey groups Test Dose Group ISIS material OS/OD No. of No. No OS/OD (μg/eye) animals 1 564426 PBS/ASO  0/150 4 2 ASO/ASO 450/450 3 3 ASO/ASO 750/750 2 4 ASO/ASO 1500/1500 1 5 664844 PBS/ASO  0/150 4 6 ASO/ASO 450/450 3 7 ASO/ASO 750/750 2 8 ASO/ASO 1500/1500 1 9 664867 PBS/ASO  0/150 4 10 ASO/ASO 450/450 3 11 ASO/ASO 750/750 2 12 ASO/ASO 1500/1500 1 13 664884 PBS/ASO  0/150 4 14 ASO/ASO 450/450 3 15 ASO/ASO 750/750 2 16 ASO/ASO 1500/1500 1 17 602881 PBS/ASO  0/400 4

Doses were administered on Day 1. Food was withheld prior to sedation. The animals were sedated with ketamine and dexdomitor for the dosing procedure. The eyes were cleansed with Betadine® and rinsed with sterile saline. Prior to the dose administration, a mydriatic (1% tropicamide) was instilled in each eye, followed by a topical anesthetic. An intravitreal injection of ASO or PBS was administered in each eye. A lid speculum was inserted to keep the lids open during the procedure and the globe was retracted. The needle was inserted through the sclera and pars plana approximately 4 mm posterior to the limbus. The needle was directed posterior to the lens into the mid vitreous. The test material was slowly injected into the mid-vitreous. Forceps were used to grasp the conjunctiva surrounding the syringe prior to needle withdrawal. Following dosing, all eyes were examined with an indirect ophthalmoscope to identify any visible post-dosing problems and confirm test material deposition. Sedation was reversed with antisedan. A topical antibiotic was dispensed onto each eye immediately following dosing and one day after dosing to prevent infection.

RNA Analysis

On day 70, eyes were collected within 10 min of exsanguination, rapidly frozen by submersion in liquid nitrogen, and placed on dry ice. Eyes were harvested from monkeys that had been treated with 150 μg or 450 μg of ISIS 564426, ISIS 664844, ISIS 664867, ISIS 664884 and 400 μg of ISIS 602881. RNA was extracted from the eye tissue for real-time PCR analysis of mRNA expression. The data from the PBS control eyes were evaluated and the average was calculated. Results are presented as percent inhibition of mRNA, relative to the PBS control, normalized to cone rod homeobox expression. A ‘0’ value inhibition only indicates that the oligonucleotide did not inhibit expression of in this particular instance.

TABLE 44 % rhodopsin inhibition compared to PBS control ISIS No Dose (μg) % inhibition 564426 150 0 450 25 664844 150 8 450 14 664867 450 21 664884 150 10 450 46 602881 400 54 Electroretinography (ERG)

The potential effect of the antisense oligonucleotides on ocular tolerability was determined by measuring the ERG response of the animals following 9 weeks of treatment. The light-adapted b-wave ERG response provided an assessment of the function of the cone photoreceptors and the bipolar cells in the eye (Hood and Birch, Visual Neuroscience. 1992. 8: 107-126; Bouskila et al., Plos One 2014. 9: e111569). Electroretinograms (ERGs) were recorded using a UTAS E-3000 Visual Electrodiagnostic System. Light-adapted b-wave ERG responses in anesthetized monkeys were measured after stimulation with white light at luminance intensity of 2.7 cd·m².

The results are presented in the Table below as percent of baseline amplitude (means z SD). As shown in the Table below, at the higher dosage of 750 μg of ISIS 564426, ISIS 664867 and ISIS 664884 per eye, the b-wave response trended towards lower levels. Furthermore, response in animals treated with ISIS 564426 trended lower at a dose of 450 μg per eye. These results indicate that ISIS 664844 is more tolerable than ISIS 564426, ISIS 664867, or ISIS 664884.

TABLE 45 Light-adapted (photopic) b-wave amplitude (% baseline) Dose Level (μg/eye) ISIS No 0 150 450 750 564426 88 ± 24 94 ± 27 50 ± 18 48 ± 19 664844 111 ± 43  87 ± 36 78 ± 13 106 ± 47  664867 83 ± 28 69 ± 14 53 ± 18 25 ± 26 664884 84 ± 7  107 ± 41  82 ± 29 35 ± 24 Pathology

After exsanguination, eyes with bulbar conjunctivae and attached optic nerve were collected from various groups and preserved in modified Davidson's fixative for 48-72 hours. The tissues were then transferred to 70% alcohol for at least 24 hours prior to processing to paraffin block. The paraffin-embedded samples were sectioned parallel to the ciliary artery to include optic nerve, macula, and optic disc. After the section was faced, 5 sections at approximately 30-micron steps, were collected. The sections were mounted on glass slides, stained with hematoxylin and eosin and analyzed for histopathology. The findings are presented in the Table below. ‘OS’ indicates ‘outer stripe’; ‘IS’ indicates ‘Inner stripe’; ‘ONL’ indicates ‘outer nuclear layer’; ‘INL’ indicates ‘inner nuclear layer’; ‘GCL’ indicates ‘ganglion cell layer’. These results indicate that ISIS 664844 is more tolerable than ISIS 564426, ISIS 664867, or ISIS 664884.

TABLE 46 Pathology findings in monkey screening study Dose/eye ISIS No 450 μg 750 μg 1500 μg 564426 Not Not Min decreased remarkable remarkable cellularity ONL 664844 Not Not Not remarkable remarkable remarkable 664867 Not Slightly decreased Slightly decreased remarkable cellularity ONL cellularity ONL 664884 Not Slightly decreased Loss of ONL, IS remarkable cellularity ONL; and OS; Slight slight vacuolation decreased ONL cellularity GCL and INL Additional Tolerability Assays

Ophthalmic examinations were conducted by an Ophthalmology Individual Scientist once during pretreatment, during week 1 (within 2-4 days following dose administration), and during weeks 3, 6, and 9. The animals were lightly sedated with ketamine prior to this procedure. Slit lamp biomicroscopy and indirect ophthalmoscopy was used. The anterior segment was scored using the Hackett McDonald scale (Hackett, R. B. and McDonald, T. O. 1996. “Assessing Ocular Irritation” in: Dermatotoxicology. 5^(th) edition. Ed. By F. B. Marzuli and H. I. Maiback. Hemisphere Publishing Corp., Washington, D.C.).

Tonometry assessments were performed once pretreatment and during weeks 3 and 9 at approximately the same time of day. Intraocular pressure (IOP) measurements were performed on sedated animals using a pneumotonometer under laboratory light conditions.

Pachymetry (corneal thickness) measurements were performed once pretreatment and during weeks 6 and 9. Measurements of the central cornea was performed on sedated animals.

Non-contact Specular Microscopy (NCSM) was performed once pretreatment and during weeks 5 and 9.

All the assessments are tabulated below. A ‘√’ sign indicates acceptable results; a ‘X’ indicates not acceptable. The results indicate that ISIS 664844 is more tolerable compared to ISIS 564426, ISIS 664867, or ISIS 664884.

TABLE 47 Tolerability screen in monkey study ISIS ISIS ISIS ISIS ISIS Test Utility 564426 664844 664867 664884 602881 Ophthalmic Cataracts, major retina ✓ ✓ ✓ X ✓ Exam or vitreous abnormalities Tonometry IOP ✓ ✓ ✓ ✓ ✓ Pachymetry Corneal thickness ✓ ✓ ✓ ✓ ✓ NCSM Corneal endothelial ✓ ✓ ✓ ✓ X cellularity, corneal thickness Histology Cellularity changes ✓ ✓ X X ✓

Example 11: Screening Summary

Over 400 antisense oligonucleotides (>200 ASOs having a MOE sugar modification and >200 ASOs having a cEt modification) were screened as described in Examples 1-10 above. Out of more than 400 ASOs, ISIS 664844 exhibited the best combination of properties in terms of potency, tolerability, and selectivity for P23H rhodopsin. 

What is claimed:
 1. A compound comprising a modified oligonucleotide consisting of 10 to 30 linked nucleosides and having a nucleobase sequence comprising at least 10 contiguous nucleobases of any of the nucleobase sequences of SEQ ID NOs: 11-64.
 2. The compound of claim 1 wherein the modified oligonucleotide has a nucleobase sequence comprising the nucleobase sequence of any one of SEQ ID NOs: 11-64.
 3. The compound of claim 2 wherein the modified oligonucleotide consists of the nucleobase sequence of any one of SEQ ID NOs: 11-64.
 4. The compound of claim 2, wherein the modified oligonucleotide comprises:  a gap segment consisting of linked deoxynucleosides;  a 5′ wing segment consisting of linked nucleosides; and  a 3′ wing segment consisting of linked nucleosides; wherein the gap segment is positioned between the 5′ wing segment and the 3′ wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.
 5. The compound of claim 4, wherein the modified oligonucleotide has a nucleobase sequence comprising any one of SEQ ID NOs: 15, 21, 29, or
 64. 6. The compound of claim 1, wherein the modified oligonucleotide comprises at least one modified internucleoside linkage, at least one modified sugar, or at least one modified nucleobase.
 7. The compound of claim 6, wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage.
 8. The compound of claim 6, wherein the modified sugar is a bicyclic sugar.
 9. The compound of claim 8, wherein the bicyclic sugar is selected from the group consisting of: 4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)2-O-2′ (ENA); and 4′-CH(CH₃)—O-2′ (cEt).
 10. The compound of claim 6, wherein the modified sugar is 2′-O-methoxyethyl.
 11. The compound of claim 6, wherein the modified nucleobase is a 5-methylcytosine.
 12. A method of treating, ameliorating, or slowing progression of retinitis pigmentosa (RP) in a subject comprising administering to the subject the compound of claim
 1. 13. The method of claim 12, wherein the retinitis pigmentosa is autosomal dominant retinitis pigmentosa (AdRP).
 14. The method of claim 13, wherein the AdRP is associated with P23H rhodopsin. 